Optimized solutions catalog.pdf · Exosomes are subsequently quantifi ed and validated for overall protein content and ... (Human being prostatic ... profi le of common exosomal markers

Catalog 2016

Exosome experts since 2007

Lyophilized Exosome Standards• Exosome Isolation Tools • Exosome Quantifi cation Kits • RNA Extraction Kits • DNA Extraction kit •

Exosome binding Antibodies • Services

Optimized solutionsfor Exosome Research

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HansaBioMed LLC

HansaBioMed LLC is a Company entirely dedicated to research and devel-opment of products in the � eld of Exosome Sciences.

Our facilities are located in Tallinn (Estonia), in the Tallinn Technology Park (Tehnopol), and in Italy at Exo-somics Siena (Siena). HansaBioMed o� ers the widest choice and selection of products dedicated to exosome research for

Life Science applications avail-able on the global market today.We are the oldest and only company focused speci� cly on Exosome isolation and analysis, producing kits that are easy to use and high quality. Some of the products available are Exo-some Standards, Exosome bind-ing Antibodies and kits for en-richment of speci� c exosome subpopulations. � e full range of our products

and services is described on this cata-logue and can be purchased directly from our on-line shop: (www.exotest.eu/online_orders/ ).

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Catalog 2016: Table of Contents

Section 1: Lyophilized Exosome Standards 3• Lyophilized Exosome Standards from human bio� uids• Lyophilized Exosome Standards from cell culture media

Section 2: Exosome Isolation Tools 15• EXO-Prep• Immunoplates• Immunobeads

Section 3: Exosome Quanti� cation kits 29• ExoTESTTM Ready to use kits• Exo-FACS Ready to use kits

Section 4: Exosome RNA Extraction kits 37• EXO-TotalRNA• TumorEXO-TotalRNA• RNA basic kit

Section 5: Exosome DNA Extraction kits 43• EXO-DNA/PS: Circulating and exosome-associated DNA from plasma and serum• EXO-DNA/UC: Circulating and exosome-associated DNA from urine and cell media

Section 6: Antibodies for Exosome Research 47• HBM-Monoclonal antibodies• HBM-Polyclonal antibodies

Section 7: Custom Services and Collaborations 59• Exosome and microvesicles puri� cation and analysis• RNA and DNA pro� ling and Next-Gen-Sequencing (NGS)• Custom products• Additional services and project collaborations• Cell fractionation and cell proliferation kits 2016

1 Lyophilized Exosome Standards

Summary Section 1Introduction 4Lyophilized Exosome Standards from body fluids or cell culture media 5Performances 7Exosome Standards and cell lysates on request 9


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IntroductionExosomes are small endo-some derived lipid nano-particles (50-120 nm) acti-vely secreted by exocytosis by most living cells. Exo-some release occurs either constitutively or upon induction, under both normal and pathological conditions, in a dynamic, regulated and functionally relevant manner. Both quantity and molecular

composition of released exosomes depend on the physiological state of the paren-tal cells.

Lyophilization is the ideal technique for preserving the long-term stability of exosomes at 4ºC. Lyophilized exosomes can be used as control standards for multiple applications including FACS, WB, ELISA and as calibration standards for quantitation of exosome-derived markers from biological samples.

Lyophilized exosomes are easy to ship and store, and are stable for over 36 months at 4°C. Purifi ed and lyophilized exosomes are obtained from a variety of biological sources: cell culture supernatant, human plasma, serum, urine and saliva. Exosomes are purifi ed following a combination of ultracentrifugation and microfi ltration steps. Exosomes are subsequently quantifi ed and validated for overall protein content and particle number by NTA (Nanoparticles Tracking Analysis) with NanoSight LM10. Lyophilization does not aff ect the stability of purifi ed exosomes and the expression levels of their exosome markers (proteins and nucleic acids).

1-Exosome Standard from Human Body Fluids Purifi ed exosomes from human Plasma, Serum and Urine (pools of healthy donors)

2-Exosome Standard from Cell Culture Supernatants Purifi ed exosomes from supernatants of a wide list of human tumor cell lines

3-Cell lysates Whole cell lysates from a wide list of human tumor cell lines

Applications:• Assay calibration

• Control (spike-in) for exosome quantifi cation

• Protein marker analysis using diff erent techniques

• Extraction and analysis of exosome nucleic acid

• Standardized positive controls for immunocapture performance evaluation

• Flow cytometry

• Electron microscopy

Advantages• Highly pure

• Easy to reconstitute

• Easy to ship and store (+4°C)

• Long term storage stability (36 months)

• Bett er performances then competitors

• Exosomes available from a large cell line bank (over 100 cell lines)

• Exosomes availabile from diff erent body fl uids (Plasma, Serum, Urine, Saliva)

1Lyophilized Exosome Standards

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Exosome Standards from Human Body Fluids and Cell Media

HansaBioMed provides lyophilized Exosome Standards from Human Plasma, Serum, Urine and Saliva pool of certificated healthy donors and from different cell lines in continous culture. All Exosome Standards are quantified and validated for overall pro-tein content and particle number by NTA with NanoSight LM10. They can be easily reconstituted by adding the appropriate volume of deionized water (MilliQ) and, once reconstitued, they can be stored at -20°C for up to 1 month and at - 80°C for up to 6 months.

Cat Code Contents Package size (#)

Lyophilized Exosome Standards from Human Biofluids

Lyophilized Exosome from Human Plasma of healthy donors

HBM-PEP100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials


Lyophilized Exosome from Human Serum of healthy donors

HBM-PES100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-PES30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosome from Human Urine of healthy donors

HBM-PEU100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials


Lyophilized Exosome from Human Saliva of healthy donors

HBM-PESL100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-PESL30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes Standards from Cell Culture Media

Lyophilized Exosomes from COLO1 cell line (Human colon carcinoma)

HBM-COLO-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-COLO-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from MM1 cell line (Human melanoma)

HBM-MM1-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-MM1-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from BLCL21 cell line (EBV transformed lymphoblastoid B cells)

HBM-BLCL-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-BLCL-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from U87 MG cell line (Human glioblastoma astrocytoma)

HBM-U87-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-U87-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from SK-N-SH cell line (Human neuroblastoma)

HBM-SK-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-SK-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from HCT116 cell line (Human colon carcinoma)

HBM-HCT-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-HCT-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Storage of Lyophilized Exosome Standards. Lyophilized Exosome Stan-dards can be stored at 4°C for up to 36 months.

Storage of reconstitud-ed exosomes.After reconstitution Exosomes can be stored at -20°C for up to one month or at -80°C for up to six months. Avoid freeze and thaw cycles.

Ordering informationsProducts can be purchased directly in our on-line shop:www.exotest.eu/online_or-ders/

US Customers:[email protected]

Distributors: www.hansabiomed.eu/index.php/distributors


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Cat Code Contents Package size (#)

Lyophilized Exosomes Standards from Cell Culture Media

Lyophilized Exosomes from PC3 cell line (Human Prostate adenocarcinoma grade IV)

HBM-PC3-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-PC3-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from BPH-1 cell line (Human being prostatic hyperplasia)

HBM-BPH-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-BPH-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from DAUDI cell line (Human Burkitt Lymphoma)

HBM-DAUDI-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-DAUDI-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from A549 cell line (Lung carcinoma)

HBM-A549-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-A549-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from K-562 cell line (Pleural effusion, leukemia chronic myelogenous)

HBM-K562-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-K562-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

Lyophilized Exosomes from B16F10 mouse cell line (mouse melanoma cell line)

HBM-B16-100/# 100 μg (>1x10^10 particles) 2 vials 4 vials 6 vials

HBM-B16-30/# 30 μg (>1x10^8 particles) 2 vials 4 vials 6 vials

HBM can provide, upon request, Exosome Standards and Cell Lysates from more than 100 different tumor cell lines.

References

Sitar, Simona, et al. “Size characterization and quantification of exosomes by asymmet-rical-flow field-flow fractionation.” Analytical chemistry (2015).

Gardiner, Chris, et al. “Measurement of refractive index by nanoparticle tracking anal-ysis reveals heterogeneity in extracellular vesicles.” Journal of extracellular vesicles 3 (2014).

Ferrante, Sarah C., et al. “Adipocyte-derived exosomal miRNAs: a novel mechanism for obesity-related disease.” Pediatric research 77.3 (2014): 447-454.

Ordering informationsProducts can be purchased directly in our on-line shop:www.exotest.eu/online_or-ders/ US Customers:[email protected]: www.hansabiomed.eu/index.php/distributors

Info and Technical sup-portFor informations and techincal support contact us at: [email protected]

Cell Lines informationsAll HansaBioMed Exosome Standards from Cell Lines are produced using Cell Lines from the Cell Bank of the Interlab Cell Line Collection of the IRCCS AUO S.Martino IST, Istituto Nazionale per la Ricerca sul Cancro, Genova,

Italy. To order Cell Lines refer

directly to:

www.iclc.it/indexpi.html.


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PerformancesLyophilization is the ideal method for preserving exosome stability.Expression of exosomal markers was assayed with diff erent techniques (WB, FACS, ELISA) and nanovesicle count was measured with NTA. Lyophilization did not substantially aff ect exosome count or biomarker expression compared to other storage methods (Fig 1, 2, 3, 4).Comparing diff erent storage methods of exosome standards (fresh vs. frozen vs lyophilized) with an anti-CD81 ELISA assay, the loss of signal compared to fresh material is minimal when using lyophilized exosomes (CV - 15%) (Fig 4).

HBM Exosome Standards are the ideal tool for your exosome studies.HBM Exosome Standars are suitable for many diff erent applications and techniques. Examples reported in fi gures show a profi le of common exosomal markers in human plasma exosomes (HBM-PEP, Fig 5) and Beta-Actin transcript amplifi ed from RNA obtained from COLO-100, MM1-100 and BLCL21-100 standards (Fig 6)

1. Western Blot comparison of exosomal markers on fresh (F), frozen (-20ºC) and lyophilized exosomes (L)

Lyophilized ExosomesFresh Exosomes

2. Comparison of exosomal markers on fresh and lyophilized exosomes.

3. Comparative Nanosight analysis of freshly purifi ed (lower pan-el) and lyophilized plasma exosomes (upper panel).

4. ExoTEST™ comparative detection of CD81 on HMB-MM1 derived exosomes:1- Fresh exosomes; 2- Frozen exosomes (-20°C); 3- Ly-ophilized exosomes

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5. Profi le of common exosomal markers in plasma exo-some standard. HBM-PEP-30 is used to quantify each tested marker

6. β-Actin transcript amplifi cation from total-RNA ex-tracted from HBM-Exosome Standard COLO-100, MM1-100, BLCL-100

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HBM Exosome Standards guarantee higher purity and better performances over com-petitors.HBM Exosome Standard from human serum (HBM-PES) were compared to human serum exosome standard from a Com-petitor. Exosomal markers CD9 and CD63 from 50 μg of both serum exosome standards were detected using an ELISA assay (fig 6). HBM Standard (50 μg) generated the highest signal, whereas the signal from Competitor Standard (50 μg) was lower than 30 μg of HBM Standard. In a second test, 50 μg of HBM and Competitor’s Standards were serially diluted to design a standard curve for exosome quantification (fig 7). The standard curve generated with HBM Standard was linear (R2 = 0,9876) within the concentration range and suitable for exosome quantification.

6. ELISA quantification of HBM Exosome Standard vs Com-petitor’s standard (human serum) for exosomal markers CD9 and CD63

7. Standard curve for exosome quantification: HBM Exo-some Standard vs. Competitor’s standard

Why to choose HBM Exosome Standards

Characteristics HansaBioMed Exosome Standard

Competitor’s Exosome Standards

Amount per vial 100 μg 50 μg

Method of isolation Ultracentrifuge Precipitation Reagent

Nanoparticles/ml (average) > 1x10^10 >1x10^6

Nanoparticles/ml in human serum exosomes (50 μg)

3,75x10^10 p/ml 2,29x10^9 p/ml

Final form Lyophilized Frozen

Storage tempera-ture 4°C (lyophilized) -20°C

Expire time 36 months 24 months

Price (1 vial) 125 € x 100 ug over 300€ x 50 ug

HBM Exosome Standards are purified using a combi-nation of ultracentrifugation and microfiltration steps. This is a tedious and time-consuming methodology but it guarantees high purity of exosomes, with low con-tamination from other microvesicles, previously elimi-nated through microfiltration and differential centrifu-gation cycles. Conversely, chemical reagents currently used for vesicle isolation precipitate both exosomes and larger vesicles, protein-protein and protein-RNA complexes, regardless of their origin. This impairs the purity of the sample. Lyophilization also allows easy and longer term storage of purified exosomes.

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Exosome Standards and Cell lysates on request

HBM can provide Purified Exosome Standard or whole Cell Lysates from the cell lines in following list.

List of available Cell Lines for Exosome Standards purification or Cell Lysates

Cell Line Name Tissue Tumor/Pathology Description

380 peripheral blood leukemia, pre-B cell

1301 blood leukemia, acute lymphoblastic, T cell

5637 bladder carcinoma

8305C thyroid carcinoma, undifferentiated

A 2058 skin melanoma, metastatic

A-172 - glioblastoma

A-204 muscle rhabdomyosarcoma

A2780 ovary adenocarcinoma

A-498 kidney adenocarcinoma

A-704 kidney adenocarcinoma

ACHN kidney adenocarcinoma

ACN - neuroblastoma

BICR 18 larynx squamous cell carcinoma

BT-474 breast carcinoma, ductal

BT-549 breast carcinoma, ductal

BV-173* peripheral blood leukemia, pre-B cell

BxPC-3 pancreas adenocarcinoma

C33A cervix carcinoma

CA46 ascitic fluid lymphoma, Burkitt

Caco-2 colon adenocarcinoma

Caki-2 kidney carcinoma

Calu-1 lung carcinoma, epidermoid, grade III

CaSki cervix cervix carcinoma, epidermoid

CFPAC-1 pancreas adenocarcinoma

CM-S/Tum bone marrow monocyte tumor

CM-S/un bone marrow monocyte tumor

COLO 205 colon colorectal adenocarcinoma

COLO 320DMF colon adenocarcinoma

COLO 699N lung, derived from pleural fluid adenocarcinoma

COLO 741 colon carcinoma, pelvic wall metastasis

COLO 800 subcutaneous nodule melanoma

COLO 853 lymph node melanoma

COLO 858 lymph node melanoma

COR-L23 lung carcinoma, large cell

Cell Lines informationsAll HansaBioMed Exosome Standards from Cell Lines are produced using Cell Lines from the Cell Bank of the Interlab Cell Line Collection of the IRCCS AUO S.Martino IST, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy. All cell lines are also available in vital form either frozen or in culture at the cell bank of the IRCCS AOU San Martino, Genova. To order Cell Lines refer directly to: www.iclc.it/indexpi.html.

Ordering InformationsExosome Standards and Cell Lysates from the available cell lines can be ordered by e-mail at the address:[email protected]

Exosome Standards Exosome Standards from cell lines on request are available only as 100 μg size, 4 or 6 vialsCatalog CodeHBM-PEC-##-100/4(6)#- Name of cell line

* Epstein-Barr virus** Transformed by Epstein-Barr virus*** type C, type B viruses


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DBTRG.05MG brain glioblastoma

DLD-1 colon adenocarcinoma

DMS-79 lung, pleural effusion carcinoma, small cell

DOHH2* - lymphoma, follicular, B cell

DU-145 prostate carcinoma

FTC-133 thyroid carcinoma, follicular

FTC-238 thyroid carcinoma, follicular

G-361 skin melanoma

GDM-1 peripheral blood leukemia, acute myelomonocytic

GF-D8 peripheral blood leukemia, acute myeloid

H9 lymphocyte lymphoma

HCT-15 colon colorectal adenocarcinoma

HCT-8 intestine, ileocecal ileocecal adenocarcinoma

HECV umbilical cord -

HEL 92.1.7 - erythroleukemia

HeLa cervix carcinoma, epitheloid

HeLa S3 cervix carcinoma, epitheloid

H-EMC-SS - chondrosarcoma

Hep G2 liver hepatocellular carcinoma

HFFF2 foreskin, fetal fibroblast, fetal

HGC-27 stomach carcinoma, undifferentiated

HL-60 peripheral blood leukemia, promyelocytic

HOS bone osteosarcoma

Hs578T breast carcinoma

Hs913T derived from metastasis to lung fibrosarcoma

HT 1197 bladder carcinoma

HT-1080 acetabulum fibrosarcoma

HT-29 colon adenocarcinoma, grade II

HuP-T3 pancreas adenocarcinoma, ascitic fluid


Hs913T derived from metastasis to lung fibrosarcoma

HT 1197 bladder carcinoma

HT-1080 acetabulum fibrosarcoma

HT-29 colon adenocarcinoma, grade II



HUVEC endothelium umbelical vein endothelial cells

IMR-32 - neuroblastoma

IMR-5 - neuroblastoma

IST-MEL1 skin melanoma



IST-MELA 16 subcutaneous metastasis melanoma, metastatic

IST-MES1 pleural effusion mesothelioma

IST-MES2 pleural effusion mesothelioma

Cell Lysates Cell Lysates from the cell line list are available in package size of 1 or 2 vials, 100 μg each one.Catalog Code:HBM- LYS-#-1(2)#- Name of cell line

Storage of Lyophilized Exosome Standards. Lyophilized Exosome Stan-dards can be stored at 4°C for up to 36 months.

Storage of reconsti-tuted exosomes.After reconstitution Exosomes can be stored at -20°C for up to one month or at -80°C for up to six months. Avoid freeze and thaw cycles.



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IST-SL1 lung carcinoma, small cell

KARPAS-422* - lymphoma, follicular, B cell

KYSE-30 oesophagus carcinoma,squamous cell

LB4** lymphocyte, B paroxysmal nocturnal hemoglobinuria

LB-B7** lymphocyte, B paroxysmal nocturnal hemoglobinuria

LB-F9** lymphocyte, B paroxysmal nocturnal hemoglobinuria

LNCap.FGC prostate adenocarcinoma

LoVo colon adenocarcinoma

LS 180 colon colorectal adenocarcinoma

M07e peripheral blood leukemia, acute megakaryoblastic

MCF7*** breast adenocarcinoma

MDA-MB-415 breast adenocarcinoma

MDA-MB-435S breast carcinoma, ductal

MDA-MB-436 mammary gland adenocarcinoma



MeCo 05 skin melanoma

MEG-01 bone marrow leukemia, megakaryoblastic

MEGR 07 metastatic cutaneous nodule melanoma

MeMo 05 lymph node, metastasis melanoma

MEMOR 06 subcutaneous metastasis malignant melanoma

MES-SA uterus sarcoma

MEWO - malignant melanoma

MG-63 - osteosarcoma

MOLT-4 peripheral blood leukemia, T cell

MONO-MAC-6 peripheral blood leukemia,acute monocytic

MPP 89 pleural effusion mesothelioma

MRC-5 lung, fetal -

MSTO-211H - mesothelioma

NCI-H1650 lung adenocarcinoma, bronchioalveolar carcnoma (smoker patient)

NCI-H1975 lung adenocarcinoma, non-small cell (non-smoker patient)

NCI-H292 lung carcinoma, mucoepidermoid

NCI-H727 lung carcinoma, non-small cell

NT2-D1 testis carcinoma, embryonal pluripotent

OCI-AML2 peripheral blood leukemia, acute myeloid

MPP 89 pleural effusion mesothelioma

MRC-5 lung, fetal -

MSTO-211H - mesothelioma

NCI-H1650 lung adenocarcinoma, bronchioalveolar carcnoma (smoker patient)

NCI-H1975 lung adenocarcinoma, non-small cell (non-smoker patient)

NCI-H292 lung carcinoma, mucoepidermoid

NCI-H727 lung carcinoma, non-small cell



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NT2-D1 testis carcinoma, embryonal pluripotent

OCI-AML2 peripheral blood leukemia, acute myeloid

PA-1 ovary teratocarcinoma

PF-382 pleural effusion leukemia, T cell

PSN1 pancreas adenocarcinoma

RAJI - lymphoma, Burkitt

Rj2.2.5 - lymphoma, Burkitt

RO82-W-1 thyroid carcinoma, follicular

ROV-S** bone marrow lymphoblastoid, EBV transformed

RPMI 7932 skin melanoma

Saos-2 bone osteosarcoma

SH-SY5Y bone marrow metastasis neuroblastoma

SiHa cervix carcinoma, squamous cell

SK-BR-3 breast adenocarcinoma

SK-HEP-1 liver adenocarcinoma

SK-LU-1 lung adenocarcinoma, grade III, poorly differenti-ated

SK-MEL-24 skin melanoma



SK-MES-1 lung carcinoma, squamous cell

SK-N-AS - neuroblastoma

SK-N-BE(2) bone marrow neuroblastoma

SK-N-BE(2)-C bone marrow neuroblastoma

SK-N-F1 bone marrow metastasis neuroblastoma

SUP-T1 pleural effusion lymphoma, lymphoblastic, T cell

SW1353 bone chondrosarcoma

SW48 colon adenocarcinoma, grade IV

SW480 colon adenocarcinoma, grade III-IV

SW620 colon adenocarcinoma, metastasis to lymph node

SW837 rectum adenocarcinoma, grade IV

T47D breast carcinoma, ductal

T84 colon carcinoma, metastasis to lung

TE671 Subline 2 - rhabdomyosarcoma

TF-1 bone marrow erythroleukemia

THP-1 peripheral blood leukemia, acute monocytic

THP-1h peripheral blood leukemia, acute monocytic

THP-1l peripheral blood leukemia, acute monocytic

U251 MG - glioblastoma-astrocytoma, grade III

U87 MG brain glioblastoma-astrocytoma

U87/DK brain glioblastoma, transfected with binase (ATP binding site 721), mutated de 2-7 EGFR

U87/WT brain glioblastoma, transfected with EGFR

U-937 pleural effusion lymphoma, histiocytic* Epstein-Barr virus** Transformed by Epstein-Barr virus*** type C, type B viruses


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VA-ES-BJ skin sarcoma, epitheloid

WiDr colon colorectal adenocarcinoma

Y79 - retinoblastoma


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Exosome Isolation Tools2Summary Section 2Introduction 16EXO-Prep 17EXO-Prep performances 18Immunoplates for Exosome capture and isolation 20Immunoplate performances 22Latex immunobeads for Exosome capture and isolation 24Latex immunobead performances 26

2 Exosome Isolation Tools

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IntroductionUltracentrifugation is currently the gold standard methodology for exosomes iso-lation from biological fl uids or cell supernatants. However it does not eff ectively isolate exosomes from small volume samples, it requires expensive equipment and it is time-consuming. To address these issues, the HBM team has developed and optimized several tools for isolating overall or specifi c exosome populations, in an effi cient, faster and cheaper way. We present three classes of products:

1-EXO-Prep New and effi cient one-step method for total exosome isolation from biofl uids (i.e. plasma, serum and urine) and from cell culture media.

2-ImmunoplatesFor exosome immunocapture using generic or specifi c exosome-associated bio-markers (i.e. tumoral, neural and glial derived).

3-ImmunobeadsLatex immunobeads for generic and specifi c exosome immunocapture from human biofl uids or cell supernatants. Immunocaptured exosome can be recovered and used for downstream analyses.


Technical supportFor informations and techincal support contact us at: [email protected]

2Exosome Isolation Tools

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EXO-PrepEXO-Prep is a fast and convenient meth-od of exosome isolation from biofl uids and cell culture supernatants. Isolation with EXO-Prep is based on chemical precipitation. Samples are incubated with EXO-Prep solution on ice, so that exosomes will precipitate following cen-trifugation. Th e obtained pellet can be resuspended in PBS 1X or deionized wa-ter. Th e protocol is user friendly, time-saving (around 1 hour), and does not require capital laboratory equipment.

Isolated exosomes are suitable for a wide range of analyses, such as NTA, protein profi ling by using diff erent techniques (western blott ing, ELISA, FACS), nucleic acid extraction and profi ling of mRNA or small RNA markers.

Cat. Code Volume Reactions

EXO-Prep for Exosome Isolation from Plasma and Serum

HBM-EXP-B5 5 ml 180 reactions Plasma, 80 reactions Serum



EXO-Prep for Exosome Isolation from Cell Media

HBM-EXP-C25 25 ml 25 reactions

HBM-EXP-C50 50 ml 50 reactions

EXO-Prep for Exosome Isolation from Urine

HBM-EXP-U25 30 ml 25 reactions

HBM-EXP-U50 60 ml 50 reactions

Applications• Single step isolation of

exosomes from biofl uids and cell supernatants.

• Isolate the overall exosome population in a sample.

• Isolated exosomes can be used for NTA analysis.

• Isolated exosomes are suitable for protein profi ling (WB, ELISA, FACS).

• Isolated exosomes are suitable for nucleic acid extraction and profi ling.

Advantages• Time and money saving.

• No ultracentrifugation required.

• Easy and fast protocol.

• Isolate exosome from small volumes of sample (as low as 100 μl of plasma).

• Easy to store and ship (4°C).


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PerformancesEXO-Prep isolates exosomes in one single step from a small volume of sampleNTA analysis (Fig 1) of plasma sample pre and post EXO-Prep treatment shows isolation of almost the entire nanoparticle population. Th e number of nanoparticles isolated by EXO-Prep (8.67x10^11) closely corresponds to the estimated num-ber present in whole plasma (8.92x10^11). Vice versa, the remaining plasma supernatant is almost completely depleted of nanoparticles (0.91x10^11). Isolated vesicles are suitable for multiple dowstream analyses, such as protein profi ling via western blott ing (Fig 2) or ELISA (Fig 3) or miRNA marker profi ling (Fig 4).

1. NTA and exosome detection in whole plasma (A), EXO-Prep isolated exosomes resuspended in 100 ul of PBS 1x (B) and plasma post EXO-Prep treatment (C).

2. Detection of exosome marker Alix in protein lysates (30 μg) from exo-somes isolated with EXO-Prep from 100 μl and 500 μl of plasma/serum and 10 and 20 ml of whole urine. Pro-tein lysates (30 μg) from exosomes purifi ed by ultracentrifugation (UC) were used as control.


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EXO-Prep guarantees exosome isolation from complex biofluids, high performances and cheaper price than competitor productsEXO-Prep is a fast and efficient method for exosome isolation from biofluids or cell culture media. In particular, when compared to a competitor product on the market, EXO-Prep allows exosome isolation from a smaller volume (100 ul) of complex biofluids as plasma, without thrombin pretreatment or difficult solubilization of exosome pellet. EXO-Prep-isolated exosomes are suitable for different analyses: protein profiling via western blotting (Fig 2) or ELISA (Fig 3), nucleic acids extraction and miRNA/mRNA profiling (Fig 4), FACS.

Characteristics EXO-Prep Competitor

Exosome isolation from cell supernatants Yes Yes

Exosome isolation from complex biofluids (plasma, serum, urine) Yes Yes, but pellet is hard to solubilize

Plasma exosome isolation

Thrombin pretreatment Not required Necessary

Pellet solubility Easy Very hard

Minimum sample amount 100 μl 250 μl

Isolated exosome are intact and suitable for ELISA assay Yes No suitable for ELISA quantification

Suitable for nucleic acids extraction and analysis Yes Yes

Price (for 5 ml of reagent) ●● ●●●

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I1 EP I2 EP I1 Comp I2 Comp UC 1 UC 2

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3. CD9 expression of plasma exosomes from two healthy individuals (100 μl and 250 μl) using EXO-Prep (EP) isolation reagent com-pared to a competitor reagent (Comp) and exosomes purified by ultracentrifugation (UC).

4. RNA extraction and profiling of 4 exosome miRNA from exosomes isolated by EXO-Prep, a Competitor reagent and ultra-centrifugation (UC). 100 μl of plasma sample have been used.


20

Immunoplates for Exosome capture and isolation

HBM Immunoplates are 96 multiwell plates covalently pre-coated with specifi c exosome-binding antibodies allowing exosome capture and isolation from dif-ferent sources (cell supernatant, human plasma, serum, urine and saliva). Covalent coating improves the correct orientation of antibodies maximizing the quantity of immunocaptured exosomes and increas-ing the binding effi ciency of the plate. In addition the coating chemistry reduces the aspecifi c binding of circulating protein complexes and cell debries, and it helps the enrichment of exosome subpopula-tions. HBM has developed diff erent types of plates for capturing the overall or en-riching specifi c exosome subpopulations (tumoral, neural, glial, monocytes and platelets). Plates are blocked and stabi-lized for long term storage.

Capture of overall ExosomesPlates are coated with antibodies against exosome surface antigens, present on overell exosome popula-tion.- From human biofl uids (plasma, serum, urine, saliva)- From cell media of human or mouse cell lines

Capture of specifi c Exosome sub-population Plates are coated with antibod-ies overexpressed in particular pathological conditions on exosome surface.

- Capture from human plasma- Capture and enrichment of tumor, glial, neuro, platelet derived exo-somes

Applications• Multiple profi ling of

exosomal markers from a single sample or screening of a large number of samples.

• Exosome capure and quantifi cation from human biofl uids (plasma, serum, urine, saliva).

• Exosome capture and quantifi cation from human or mouse cell media.

• Titration of purifi ed exosomes.

• Capture and enrichment of specifi c exosome subpopulations.

Advantages• Ready to use.

• Long term storage (up to 2 years).

• No exosome pre-purifi cation required (by ultracentrifugation or other methods).

• Small amount of sample required (100 μl per well).

• Suitable for nucleic acid extraction from exosome captured on the plate.

• Flexibility in designing a multiplexing assay.

• Open platf orm for customized coating solutions .


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Immunoplates for overall Exosome capture

Cat. Code Type of immunoplate Package size Coating antibody

Overall Exosome capture from human plasma and urine

HBM-POF-CC/T1 or T5 Transparent 1 or 5 plates Rabbit

HBM-POF-CC/W1 or W5 White 1 or 5 plates Rabbit

HBM-POF-CC/B1 or B5 Black 1 or 5 plates Rabbit

Overall Exosome capture from human serum

HBM-POS-CC/T1 or T5 Transparent 1 or 5 plates Mouse

HBM-POS-CC/W1 or W5 White 1 or 5 plates Mouse

HBM-POS-CC/B1 or B5 Black 1 or 5 plates Mouse

Overall Exosome capture from human saliva

HBM-POSL-CC/T1 or T5 Transparent 1 or 5 plates Rabbit

HBM-POSL-CC/W1 or W5 White 1 or 5 plates Rabbit

HBM-POSL-CC/B1 or B5 Black 1 or 5 plates Rabbit

Overall Exosome capture from cell media

HBM-POC-CC/T1 or T5 Transparent 1 or 5 plates Mouse

HBM-POC-CC/W1 or W5 White 1 or 5 plates Mouse

HBM-POC-CC/B1 or B5 Black 1 or 5 plates Mouse

Immunoplates for capture of specific Exosome sub-populations

Cat. Code Type of immunoplate Package size Coating antibody

Tumor-derived Exosome capture and enrichment from human plasma

HBM-PTF-CC/T1 or T5 Transparent 1 or 5 plates Rabbit

HBM-PTF-CC/W1 or W5 White 1 or 5 plates Rabbit

HBM-PTF-CC/B1 or B5 Black 1 or 5 plates Rabbit

Neural-derived Exosome capture and enrichment from human plasma

HBM-PNF-CC/T1 or T5 Transparent 1 or 5 plates Rabbit

HBM-PNF-CC/W1 or W5 White 1 or 5 plates Rabbit

HBM-PNF-CC/B1 or B5 Black 1 or 5 plates Rabbit

Glial-derived Exosome capture and enrichment from human plasma

HBM-PGF-CC/T1 or T5 Transparent 1 or 5 plates Mouse

HBM-PGF-CC/W1 or W5 White 1 or 5 plates Mouse

HBM-PGF-CC/B1 or B5 Black 1 or 5 plates Mouse

Monocytes/Platelet-derived Exosome capture and enrichment from human plasma

HBM-PPP-CC/T1 or T5 Transparent 1 or 5 plates Mouse

HBM-PPP-CC/W1 or W5 White 1 or 5 plates Mouse

HBM-PPP-CC/B1 or B5 Black 1 or 5 plates Mouse

Type of ImmunoplateDifferent plates are available for colorimetric, luminometric or fuorimetric readings (transparent, white and black, respectively).

Storage conditionsUnopened: 2 years, stored at 4°C.

Opened: 6 month stored at 4°C.

Amount of material to useIt is recommended to load 100 μl of sample per well. Whole human plasma and serum can be loaded for exosome capture.

Using urine and cell media, it is recommended to concentrate the sample 10X, in according to our protocol, before to load the sample on the plate.

Packaging Immunoplates are individually sealed in an opaque aluminium ziplock bag, compliant to pharmaceutical regulations. Easy to open and resealable.


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PerformancesImmunoplates allow exosome protein profiling without pre-purification (by ultracentri-fuge, chemical precipitation or microfiltration).HBM plates are useful tools for immunocapturing exosomes from biofluids or culture media, for protein analyses and pro-tein marker profiling. They allow quantitative and qualitative simultaneous analysis of different protein markers (Fig 5) from the same sample, or expression profiling of a single marker in different samples (Fig 6), without exosome pre-purification via ultracentrifuge or other methods. CD9 expression (Fig 7) of immunocaptured plasma exosomes mimic that of the cor-respondent ultracentrifuged fraction. No significant cross-reactivity is observed with soluble antigens or other vesicle-associated proteins (Fig 8).

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7. Comparison of CD9 detection on purified (ultracentrifuged) plasma exosomes vs unfractionated samples in a set of healthy donor’s plasma

8. HBM plate is selective in capturing purified exosomes (pel-let after centrifugation 120000g) and no other circulating mi-crovesicle (pellet 1200g and 10000g)

5. Common exosomal biomarkers analysis in a healthy donor’s plasma sample

6. CD63 profiling on exosomes derived from supernatants of different cell lines


23

Immunoplates enable enrichment in tumor-derived exosome subpopulation from hu-man plasmaTumor-derived Exosome capture Immunoplates are precoated with a proprietary capturing antibody which allows enrich-ment and selection of exosomes from tumor origin from human plasma, particularly useful for cancer biomarker profiling (Fig 9). Figure 10 shows that by simply using the Tumor-derived exosome plate it is possible to discriminate healthy donors from cancer patient (indicated by a black arrows).

Specific Immunoplates for enrichment in neural, glial, platelet-derived exosome sub-population from human plasma

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9. Enrichment in Tumor-derived exosome in early and late stage melanoma (Mel E; Mel ADV) and ovary carci-noma (Ova E, Ova ADV) using Tumor-derived exosome capture plate vs Overall exosome capture plate (blank subtracted).

Immunoplate for Tumor-derived exosome capture Immunoplate for overall exosome capture

10: Immunoplate for Tumor-derived exosomes capture allows dis-crimination of cancer patients (black arrows)

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11. Specific immunocapture of 30 μg of exosome isolated from Neuroblastoma (SH-30) or Glioblastoma (U87-30) cell lines in specific immunoplates. COLO cell and plasma (PEP) purified exosomes were used as controls.

These plates are precoated with exosome associated antibody in-dicative of neurological, glial or monocyte/platelet origin enabling specific enrichment of exosomes from human plasma samples. In the examples reported (Fig 11) we show the specificity of bind-ing of exosome derived from neuroblastoma (SH) or glioblastoma (U87) cell lines when plates for neural or glial-derived exosome capture are used. In figure 12 and 13, increasing amount of puri-fied exosomes from neuroblastoma (SH) or glioblastoma (U87) cell lines were spiked in human plasma from healthy donors. Selective capture of specific exosome subpopulation was tested comparing the same plasma sample after spiking of purified plasma exosomes (HD). In both experiments (Fig 12, 13) the signal relative to the specific exosomes subpopulation increases after spiking, while no changes are detectable with HD, suggesting the enrichment of neural or glial derived exosomes respectively.

12. Immunocapture of neuroblastoma derived exosomes (SH) subpopulation diluted in human plasma from healthy donors.

13. Immunocapture of glioblastoma derived exosomes (U87) subpopulation diluted in human plasma from healthy donors.


24

Latex Immunobeads for Exosome capture and isolation

HBM provides several types of Immunobeads for captur-ing and isolating overall or specifi c exosome sub-popu-lations. Latex immunobeads are covalently coupled with antibodies against exosome surface antigens, allowing exosome capture from hu-man biofl uids (tested for plasma, serum and urine) and cell culture media without

pre-purifi cation steps (ultracentrifuge or other method for exosome purifi cation). HBM immunobeads are able to capture the overall exosome population (Immuno-beads for Overall Exosome capture) or to enrich exosome subpopulation derived from tumoral source (Tumoral-derived exosome capture and enrichment). Immu-nobeads are supplied with an Exosome Elution Buff er, that allows detachment and elution of captured exosomes for downstream analyses, and with a Beads Regenera-tion Buff er to regenerate immunobeads for further usage. All latex Immunobeads are available in two sizes (0.4 and 1 micron of diameter) and are sold in packages of 10 and 20 reactions, or as TEST format including material for 3 and 5 reactions.

Beads are mixed together with the sample and incubated over night (ON) for exo-somes binding. Aft er ON incubation beads can be easily recovered by centrifu-gation and washed with appropriate buff er. Bead pellet can be directly used for nucleic acid extraction or protein analysis. Alternatively exosomes can be eluted off the beads with the Exosome Elution Buff er and used for functional studies.

Applications• Overall exosome isolation

from cell culture media and human biofl uids (tested for plasma, serum, urine).

• Overall exosome isolation from mouse biofl uids (tested for plasma and serum).

• Capture and enrichment of human exosome subpopulation (tumor-derived).

• Downstream exosome marker profi ling.

• Nucleic acids extraction

• Exosome elution from immunobeads.


• Easy, fast and effi cient protocol.

• Small sample volume of biofl uid or cell culture medium.

• No ultracentrifugation or other methods for exosome purifi cation required.

• Supplied with buff er for exosome elution from beads.

• Immunobeads can be regenerated with Beads Regeneration Buff er and reused.


25

Latex Immunobeads for Exosome capture

Cat. Code Immunobead diameter Package Coating antibody

Overall Exosome immucapture from human biofluids

HBM-BOLF-CC/10-04 0.4 micron 10 reactions Mouse

HBM-BOLF-CC/10-1 1 micron 20 reactions Mouse

HBM-BOLF-CC/20-04 0.4 micron 10 reactions Mouse

HBM-BOLF-CC/20-1 1 micron 20 reactions Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions. Cat Code: HBM-TBOLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

Overall Exosome immunocapture from cell culture media

HBM-BOLC-CC/10-04 0.4 micron 10 reactions Mouse

HBM-BOLC-CC/10-1 1 micron 20 reactions Mouse

HBM-BOLC-CC/20-04 0.4 micron 10 reactions Mouse

HBM-BOLC-CC/20-1 1 micron 20 reactions Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions. Cat Code: HBM-TBOLC-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

Tumor-derived Exosome immunocapture from human biofluids

HBM-BTLF-CC/10-04 0.4 micron 10 reactions Rabbit

HBM-BTLF-CC/10-1 1 micron 20 reactions Rabbit

HBM-BTLF-CC/20-04 0.4 micron 10 reactions Rabbit

HBM-BTLF-CC/20-1 1 micron 20 reactions Rabbit

Immunobeads are also available in TEST format for 3 and 5 reactions. Cat Code: HBM-TBTLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

Overall Exosome immunocapture from mouse plasma and serum

HBM-BMLF-CC/10-04 0.4 micron 10 reactions Mouse

HBM-BMLF-CC/10-1 1 micron 20 reactions Mouse

HBM-BMLF-CC/20-04 0.4 micron 10 reactions Mouse

HBM-BMLF-CC/20-1 1 micron 20 reactions Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions. Cat Code: HBM-TBMLF-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

Overall Exosome immunocapture from mouse cell media

HBM-BMLC-CC/10-04 0.4 micron 10 reactions Mouse

HBM-BMLC-CC/10-1 1 micron 20 reactions Mouse

HBM-BMLC-CC/20-04 0.4 micron 10 reactions Mouse

HBM-BMLC-CC/20-1 1 micron 20 reactions Mouse

Immunobeads are also available in TEST format for 3 and 5 reactions. Cat Code: HBM-TBMLC-CC/# (3 or 5 reactions)-## (bead diameter, 0.4 μm or 1 μm)

Storage conditionsImmunobeads must be stored at 4°C.

Amount of material to useRecommended starting volume from 0.1 ml - 0.5 ml of plasma, from 0.5 ml to 1 ml of serum.

Concentrated (10X) urine and cell culture medium samples are recommended prior capture according to our suggested protocol.

Packaging Immunobeads (10 and 20 reaction packages) are supplied with Exosome Elution Buffer, for eluting intact exosomes from beads and with Bead Regeneration Buffer, for regenerating immunobeads that can be reused at least once more.


26

PerformancesImmunobeads are an useful tool for exosome protein profi lingFollowing incubation, beads were recovered by centrifugation, resuspended in Laemmli buff er, and loaded on polyacryl-amide gel for western blott ing analysis. Exosome were detected with an anti-Alix antibody (human plasma and cell super-natant as described in fi g 14 and 15 respectively).

Immunobeads enrich for Tumor-derived exosome subpopulation in cancer patientsImmunobeads for tumor-derived exosome capture can be used for enriching exosome subpopulations derived from tumoral sources thus providing a novel platf orm for cancer biomarker research.

14. Alix expression by western blott ing of human plasma exo-somes captured on HBM-Immunobeads from 0.5 ml of plasma in comparison with exosomes purifi ed via ultracentrifuge. 1, 3 and 5: Immunocapture with immunobeads from 1 ml, 0.5 ml and 100 μl of human plasma respectively. 2, 4, 6: Ultracentrifuged exosomes from plasma after immunobeads capture. 7 Immunocapture with immunobeads isotype coupled. 8, 9: Ultracentifuged exosome from 0.5 ml and 1 ml of human plasma.

15. Alix expression by western blott ing of exosomes captured on HBM-BOLC immunobeads from COLO1 cell supernatant vs iso-type coupled beads.

1, 3 and 5: Immunocapture with immunobeads from 1 ml, 0.5 ml and 100 μl of human plasma respectively. 2, 4, 6: Ultracentrifuged exosomes from plasma after immunobeads cap-ture. 7 Immunocapture with immunobeads isotype coupled. 8, 9: Ultracentifuged exosome from 0.5 ml and 1 ml of human plasma.

COLO1 Cell Medium

HBM-BOLC immunobeads

Isotypecoupled

Immunobeads for Overall Exosome capture

Immunobeads for Tumor derived exosome capture

16. Anti-Alix WB analysis on exosomes immunocaptured with beads for overall (HBM-BOLF) and for tumor-derived exosomes (HBM-BTLF). WB shows the capture of exosomes only for cancer patients when beads for tumor-derived exosomes are used.


27

Latex Immunobeads are suitable for exosome RNA extraction and RNA marker profi lingImmunobeads allow total RNA extraction from captured exosomes. Isolated total RNA is suitable for mRNA or small RNA analysis. In fi gure 17 is shown the expression profi le of TM9SF4 transcript in cancer cell derived exosomes.

Exosomes can be succesfully eluted from immunobeads which can be reused twice moreExosomes immunocaptured from 1ml of human plasma using latex beads were eluted with Exosome Elution Buff er and then quantifi ed onto HBM immunoplate for overall exosome capture from plasma (Fig 18). Detection was performed us-ing a proprietary anti-CD9-Biotin conjugated antibody. Eluted beads can be regenerated with Bead Regeneration Buff er and reused for capturing exosome two more times (Fig 19).

17. Profi le of two mRNA marker in exo-somes derived from 9 cancer cell lines, previously immunocaptured using HBM beads.

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28

Exosome Quantification Kits3Summary Section 3Introduction 30ExoTESTTM Ready to Use ELISA quantification kit 31ExoTESTTM performances 33Exo-FACS Ready to Use kit 35Exo-FACS performances 36

3 Exosome Quantifi cation Kits

30

IntroductionExosome research is a relatively novel and exciting fi eld of biomedical research with development of applications in vaccines and in cancer. Th e use of exosomes for as-sessment of functional state of parent cell in basic and clinical research is currently hampered by the lack of reliable, aff ordable and optimized tools for quantitative analysis of exosomes in complex biological samples (e.g. plasma or urine). In this section we present ExoTESTTM an innovative technology that allows purifi cation, quantifi cation and characterization of circulating exosomes specifi c to a cell type or tissue, or to a condition (health vs. disease). Together with ExoTESTTM HBM team has developed a new kit for FACS (Fluorescence Activated Cell Sorting) analysis of exosome markers, Exo-FACS, which allows exosome FACS profi ling without wasting time in exosome isolation by ultracentrifugation.

1- ExoTESTTM Ready to use kit: ELISA assay for exosome immunocapture, quantifi cation and characterization from human biofl uids (plasma, serum, urine, saliva) and cell culture media.

2- Exo-FACS Ready to use kit Ready to use kit which allows exosome isolation from biofl uids or cell media and biomarker analysis via FACS.



3Exosome Quantifi cation Kits

31

ExoTESTTM Ready to useELISA Quantifi cation kits

ExoTESTTM is a patented double sandwich ELISA assay for quantitative and qualita-tive analysis of exosomes. In particular ExoTESTTM is a successful platf orm for exo-some quantifi cation and characterization from small amount of human biological fl uids or cell media and it may be exploited to identify exosomes released by cancer cells in the plasma and urine of tumor patients in various disease conditions.

ExoTESTTM consists in ELISA plates pre-coated with proprietary pan-exosome antibodies enabling specifi c capture of exosomes from diff erent biological samples, including cell culture supernatants and human biological fl uids. Quantifi cation and characterization of exosomal proteins is subsequently performed using appropriate detection antibodies against exosome associated antigens that can be for either generic or cell/tissue-specifi c exosomes. Lyophilized Exosome Standards, characterized for protein content and particle number (NTA) allow the quantifi cation of unknown sample by a standard calibration curve.

Kit components Description

Immunoplate 96 well (12 strips x 8 wells) precoated with specifi c exosome capturing antibody.

Lyophilized Exosome Standards

Exosome Standards from human plasma, serum, urine, saliva or cell medium for calibration curve.

Antibodies for exosome marker detection

Primary anti-human CD9 antibody (HBM proprietary) and secondary antibody HRP conjugated for exosome detection.

Buff ers Sample buff er for antibody dilution and incubation. Washing buff er for washing ELISA plate.

Reagents Reagents for signal detection.

Applications• Exosome capture and

quantifi cation from human biofl uids and cell culture media.

• Exosome comprehensive profi ling.

• Pre-clinical research on non-invasive biomarkers for detection and monitoring of a number of pathological conditions (infl ammation, cancer, neurodegeneration, etc).


• No initial exosome purifi cation required.

• User friendly and suitable for multiple marker analyses.

• Available in TEST format (limited to 3 ELISA strips, 24 wells).

3 Exosome Quantification Kits

32

ExoTESTTM Ready to useELISA Quantification Kits

HBM offers different types of ExoTESTTM kit for quantification of Overall Exosome population from human biofluids (plasma, urine, serum) and from cell culture super-natants. In addition is available the ExoTESTTM for Tumor-derived Exosome Enrich-ment and quantification. This kit includes an immunoplate able to enrich the tumor exosome subpopulation and is especially suitable for biomarker discovery in clinical cancer research or in development of diagnostics. All ExoTESTTM kits are available with different immunoplate format for colorimetric (transparent) or luminometric (white) or fluorimetric (black) methodology.

Cat. Code Description Immunoplate

ExoTESTTM for Overall Exosome immunocapture and quantification from human plasma and urine

HBM-RTK-POF/## Exosome detection performed with anti-human CD9 antibody (HBM proprietary). Secondary antibody: anti-mouse HRP conjugated.

Transparent

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ExoTESTTM for Overall Exosome immunocapture and quantification from human serum

HBM-RTK-POS/## Exosome detection performed with anti-human CD9 antibody biotin conjugated (HBM proprietary). Signal detected with Streptavidin-HRP conjugated.

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ExoTESTTM for Overall Exosome immunocapture and quantification from cell culture media

HBM-RTK-POC/## Exosome detection performed with anti-human CD9 antibody biotin conjugated (HBM proprietary). Signal detected with Streptavidin-HRP conjugated.

Transparent

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ExoTESTTM for Tumor-derived Exosome immunocapture and quantification from human plasma

HBM-RTK-PTF/## Exosome detection performed with anti-human CD9 antibody (HBM proprietary). Secondary antibody: anti-mouse HRP conjugated.

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Custom made ExoTESTTM for Specific Exosome immunocapture and quantification

HBM-RTK-CMK HansaBioMed offers the flexibility of creating and designing your own kit by choosing among a wide variety of reagents available in our catalog. For information contact [email protected]

All kits are also available in TEST format, limited to 3 ELISA strips (24 wells). Cat Code: HBM-TRTK-###

ReferencesTetta, Ciro, et al. “Extracellular vesicles as an emerging mechanism of cell-to-cell com-munication.” Endocrine 44.1 (2013): 11-19.

Zarovni, Natasa, et al. “Integrated isolation and quantitative analysis of exosome shut-tled proteins and nucleic acids using immunocapture approaches.” Methods (2015).

Vishnubhatla, Indira, et al. “The Development of Stem Cell–Derived Exosomes As a Cell-Free Regenerative Medicine.” J. Circ. Biomark 143 (2014): 2.

Logozzi, Mariantonia, et al. “High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.” PloS one 4.4 (2009): e5219.

Amount of material to use: Starting material: 100 μl of biological sample. Whole plasma and serum can be directly loaded on the plate. It is recommeded to use concentrate (10X) urine or cell media prior capture according to our suggested protocol.

Lower detection limit:The detection limit of the assay is lower than 0.35 μg of overall exosomes which is the equivalent to less than 50 pg of targeted exosome protein.

Assay calibration:Kit contains Lyophilized Exosome Standards for assay calibration.

Storage conditions:All reagents are shipped and can be stored at 4°C as described in the datasheet of the product.


33

PerformancesExoTESTTM presents high sensitivity in detecting low exosome amount, high affi nity and low background for exosome binding and quantifi cation.Th e sensitivity of the ExoTEST™ was compared to Western blot. Th e sensitivity of the ExoTEST™ is higher than Western blott ing as reported in Figures 1 and 2 showing that 10 μg of lyophilized exosomes are equivalent to 0.1 ng of recombinant exosomal protein. Since the standard curve’s lower concentration is 0.39 μg of lyophilized exosomes (Fig 1), the sensitivity of our test is around 39 pg of protein equivalent. In addition, immunoplates used for exosome capture shows a low back-ground which makes it a reliable, effi cient and accurate test (Fig 1).

ExoTESTTM is a sensitive method for exosome quantifi cation in human biofl uids.ExoTEST enables reliable and precise quantitative measurement and comparison among samples and individual experi-ments. Standard exosome preparations are provided for designing standard curves and assay calibration. ExoTEST ensures concentration-dependent detection of exosomal markers from diff erent biological samples including human biofl uids such as plasma and urine. Th is assay provides increased sensitivity in detection of exosomal markers with respect to other ana-lytical methods (i.e FACS, WB) We report an example of exosome quantifi cation performed in 5 unknown plasma samples from healthy donors using the ExoTESTTM Ready to use kit for Overall Exosome quantifi cation from human plasma (HBM-RTK-POF/TP). Following Lyophilized Standards and unknown samples binding onto the ELISA plate, test is run in according to the kit protocol and exosome detection is performed with anti-CD9 antibody (HBM).

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1. CD9 titration (blue line) of plasma healthy donor exosome standards (HBM-PEP100) and comparison with observed back-ground (red line, only secondary antibody).

2. CD9 exosome marker detection by Western Blot-ting on lyophilized exosomes from human plasma (HBM-PEP100) and recombinant CD9 protein.

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4. CD9 titration of exosomes in 5 diff erent whole plasma from healthy donor samples.

3 Exosome Quantification Kits

34

Exosome quantification is finally performed calculating the quantity of exosomes (expressed in μg) into the 5 unknown sam-ples through the equation of the standard curve (Fig 3). The particles number contained in 100 μl of plasma is calculated from quantity of exosomes (expressed in μg) in according to the particles concentration (number of particles/ml) indicated in the label of the Lyophilized Exosome Standards (HBM-PEP100, NTA: 3x10^11 particles/ml).

Plasma sample O.D. 450 nm Exosome μg Particle number in 100 μl of plasma

#1 0,5673 12,869 3,86x10^9

#2 0,6194 14,205 4,26x10^9

#3 0,4425 9,6692 2,90x10^9

#4 0,3100 6,2717 1,88x10^9

#5 0,7853 18,458 5,54x10^9


35

Exo-FACS Ready to use kitsExo-FACS allows exosome isolation from biofl uids or cell culture media and FACS analysis of exosome markers. Th e kit consists of EXO-Prep reagent, for exosome isolation, 4 µm beads used for the overall capture of pre-isolated exosomes, lyophilized exosomes from cell culture supernatants or human biological fl uids, as positive control. Th e characterization of exosomal proteins (membrane expressed or internal) is subsequently performed using appropriate detection antibodies against exosome associated antigens.

HBM off ers diff erent Exo-FACS kits for staining of exosomal markers from human biofl uids (plasma, urine, serum, saliva) and from cell culture supernatants. Exo-FACS contains reagents for 20 reactions (lyophilized exosomes, beads, antibodies and buf-fers). Primary antibody included in the kit is against a common exosomal marker (CD9 or CD63) and can be used as a positive control for protein profi ling via FACS analysis.

Cat. Code Description Lyophilized Exosome Standard

Detection antibody

Exo-FACS ready to use kits for analysis of exosome marker from human biofl uids

HBM-FACS-PEP FACS analysis of plasma exosomes

HBM-PEP100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PES FACS analysis of serum exosomes

HBM-PES100 1 vial, 100 μg

Anti human CD9

HBM-FACS-PEU FACS analysis of urine exosomes


Anti human CD9

HBM-FACS-PESL FACS analysis of saliva exosomes


Anti human CD9

Exo-FACS ready to use kits for analysis of exosome marker from cell culture media

HBM-FACS-C FACS analysis of cell derived exosomes

HBM-###100 *1 vial, 100 μg

Anti human CD63

* Possibility to choose the Exosome Standard from the list of Lyophilized Exosome Standards from cell media available in the section 1 of this catalog, page 5 and 6.

Applications• Exosome isolation

and exosome marker characterization via FACS.

• Exosome comprehensive profi ling.


• No initial exosome purifi cation required.

• Lyophilized Exosome Standards for positive control included

• User friendly and suitable for multiple marker analyses.

Kit components• EXO-Prep, for exosome

isolation.

• Lyophilized Exosome Standards, for positive control.

• Primary antibody for exosome marker detection as positive control.

• Secondary antibody Alexa 488.

• Sample buff er, for antibody incubation.

StorageAll reagents are shipped and must be stored a 4°C

3 Exosome Quantifi cation Kits

36

PerformancesExo-FACS is an useful tool for exosome protein profi ling by using FACS techniqueExo-FACS was used for a protein marker profi le in exosomes derived from diff erent sources. Exosome binding on FACS-beads was performed by incubation at 4°C over night. Exosome-bead complex is ready to be labeled with fl uorophore-conjugated antibodies for specifi c exosome markers. In fi gure 5 is shown shown a profi le of expression of three diff erent exosome markers in exosomes purifi ed from Melanoma (MM1), Neuroblastoma (SH) and Glioblastoma (U87) cell super-natants.

5. FACS profi ling of exosomal markers CD9, CD63 and TM9SF4 in purifi ed exo-somes from MM1, SH-SY5Y and U87 cell lines.

Exosome RNA Extraction Kits4Summary Section 4Introduction 38HBM Exosome RNA extraction kits 39Performances 41

4 Exosome RNA Extraction Kits

38

IntroductionExosomes shutt le functional RNA molecules in the target cell. Increasing evidence suggests a role for exosome-derived miRNAs in the development and/or progres-sion of specifi c human diseases. Pathogenic miRNAs might be exploited as novel therapeutic targets or disease biomarkers in complex diseases, including cancer. In fact, miRNAs seem to play critical roles as transcriptional and post-transcriptional regulators of epigenetic mechanisms and cell processes and have been linked to the etiology, progression and prognosis of cancer. Similar miRNA expression patt erns between tumor tissue samples and circulating exosomes have been observed.

HBM has developed optimized solu-tions for the effi cient extraction of high-quality total RNA (miRNA and mRNAs) from the overall exosomes and microvesicle population or from tumor-specifi c exosome subpopu-lation, which helps to facilitate the identifi cation of tumor miRNA or mRNAs signatures from human bio-fl uids or cell culture media.

HBM off ers three diff erent kits for exosome capture and RNA extraction.

1- EXO-TotalRNA: Overall Exosome Immunocapture and RNA extraction kit: Kit allows, by immunocapture, the isolation of overall exosome population from hu-man biofl uids or cell culture media and RNA extraction.

2- TumorEXO-TotalRNA: Tumor Exosome Immunocapture and RNA extraction kit: Kit allows, by immunocapture, the enrichment of tumor-specifi c exosome popula-tion from human biofl uids and RNA extraction.

3- RNA Basic Kit:Kit allows RNA extraction from exosomes pre-isolated with diff erent methods (ultra-centrifugation, chemical precipitation, immunocapture, size-chromatography etc.)



4Exosome RNA Extraction Kits

39

HBM Exosome RNA extraction kitsHBM immunobeads can capture the overall exosome population from a wide range of media, including cell culture supernatants and human biofl uids (plasma, serum, urine, etc.) as well as enrich some specifi c exosome subpopulations (tumor-derived exosomes). Th e captured exosomes are subsequently lysed with an optimized lysis buff er and total RNA is purifi ed using spin columns with a fast and user-friendly process. Eluted RNA can be used for downstream analyses or stored at -80°C. Exosome standards for positive control are also included in the kit. All our kits guarantee high specifi city for exosomal RNA and high yield of total RNA (including small RNAs).

Cat. Code Description Size

EXO-TotalRNAOverall Exosome Immunocapture and RNA extraction Kit (from human biofl uids and cell media)

HBM-RNA-BOF-10/# RNA extraction from overall exosome population captured with immunobeads

10 reactions

HBM-RNA-BOF-20/# 20 reactions

TumorEXO-Total RNATumor-derived Exosome Immunocapture and RNA extraction Kit (from human biofl uids)

HBM-RNA-BTF-10/# RNA extraction from tumor-derived exosome population captured with immunobeads

10 reactions

HBM-RNA-BTF-20/# 20 reactions

RNA Basic Kit*

HBM-RNA-B20 RNA extraction from pre-isolated exosome 20 reactions

HBM-RNA-B30 30 reactions

*RNA basic kit does not contain immunobeads for exosome isolation. It is designed for exosome total RNA (miRNA + mRNAs) from pre-isolated exosomes. Compatible with exosome isolated via ultracen-trifuge, chemical precipitation, size chromatography, immunocapture etc.

Applications• Direct capture and

exosome RNA extraction from human biofl uids and cell culture media without initial exosome purifi cation step.

• Simultaneous miRNA and mRNA profi ling (qRT-PCR, RT-PCR, microarray).

• Enrichment of tumor-derived exosomes for cancer miRNA marker profi ling.

Advantages• High yield of total RNA

(including small RNAs).

• Fast and user friendly protocol.

• Small starting amount of sample (less than 1 ml).

• Bett er RNA yield than similar products.

• Th e only kit on the market providing Exosome Standards as control.


40


EXO-TotalRNA Overall Exosome Immunocapture and RNA extraction Kit (from human biofluids and cell media)

Immunobeads Immunobeads for overall exosome capture from human biofluids or cell culture media.

Beads Washing buffer (5X) Buffer for washing Immunobeads after exosome capture.

Lysis buffer Buffer for exosome lysis.

RNA Washing buffer Buffer for RNA washing in spin column.

Elution Buffer Buffer for RNA elution from spin column.

Columns and Tubes Spin columns for RNA purification and tubes for RNA collection.

Lyophilized Exosome Standard

Exosome standards (1 vial, 100 μg) that can be choosen from the list of available Lyophilized Exosome Standards (Pag 3).

TumorEXO-Total RNA Tumor-derived Exosome Immunocapture and RNA extraction Kit

Immunobeads Immunobeads for tumor-derived exosome capture and enrichment from human biofluids.

Beads Washing buffer (5X) Buffer for washing Immunobeads after exosome capture.







RNA Basic Kit





Amount of starting materialWhole human plasma and serum can be directly used for RNA extraction. Minimum volume required is 100 μl for plasma and 500 μl for serum loaded for exosome capture.

Using urine and cell media, it is recommended to concentrate the sample 10X, in according to our protocol, before to incubate the samples with immunobeads.

Shipment and storageKit is shipped a 4°C and all reagents must be stored at 4°C.

4Exosome RNA Extraction Kits

41

PerformancesEXO-Total RNA allows extraction of high quality of exosome-derived RNAs from low volumes of sample and bett er performance than competitors.Effi ciency of EXO-TotalRNA kit was tested vs a competitor kit for RNA extraction from plasma derived exosomes. Extraction of total exosome RNA was performed from 100 μl of healthy donor plasma (HD #1 an #2) with Competitor Kit or with the EXO-TotalRNA Kit (HBM). RNA quality was evaluated by electopherogram (Fig 1) with Small RNA microfl uidic chips (Agilent 2100 Bioanalyzer). RNA yield was quantifi ed by Nanodrop (Fig 2) and extracted RNA was subsequently retrotran-scribed using the miScript II RT kit (Qiagen). miR-21 and b-actin markers were amplifi ed by qPCR (Fig 3 and 4).

1. Electropherograms of small RNA extracted with HBM EXO-Total RNA kit and Competitor (Agilent 2100 Bio-anlyzer)

HD#1 HD#2

EXO-Total RNA Kit(HBM)

Competitor Kit

0

100

200

300

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600

HBM Competitor

RNA n

g

Total RNA yield (ng)24

25

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28HBM Competitor

Ct Va

lues

PCR amplification miRNA21

2. Nanodrop quantifi cation of total RNA yield

3. miRNA 21 amplifi cation by qPCR 4. β-Actin amplifi cation by PCR


42

TumorEXO Total RNA allows enrichment of cancer derived miRNA from plasma sam-ples.Total Exosome derived RNA was extracted from 100 μl of plasma from a healthy donor and a cancer patient using RNA kit EXO-Total RNA (HBM-RNA-BOF) for immunocapture of overall exosomes and RNA extraction or TumorEXO Total RNA (HBM-RNA-BTF), for tumor-derived exosome enrichment and RNA extraction, respectively. Total RNA extracted was ret-rotranscribed using the miScript II RT kit (Qiagen) and the expression level of miR-21 and miR-16 were measured relative to control miR451 by qPCR. Usage of TumorEXO Total RNA shows enrichment in miR-21 and miR-16 in cancer patient (Fig 5 and 6).

0

1

2

3

4

5

6

7

No Cancer Cancer

Fold

chan

ge (n

orm

alize

d to

miR

-451

)

miR-21 HBM-RNA-BOF

HBM-RNA-BTF

0

1

2

3

4

5

6

7

No Cancer Cancer

Fold

Cha

nge

(nor

mali

zed

to m

iR-4

51)

miR-16 HBM-RNA.BOF

HBM-RNA-BTF

5. Enrichment in miR-21 expression level in cancer when Tumor-EXO Total RNA kit is used.

6. Enrichment in miR-16 expression level in cancer when Tumor-EXO Total RNA kit is used.

Exosome DNA Extraction Kits5Summary Section 5Introduction 44EXO-DNA: Isolation of circulating and Exosome-related DNA 45Performances 41

5 Exosome DNA Extraction Kits

44

IntroductionTogether with RNAs, genomic single or double-stranded DNA and mito-chondrial DNA have been recently de-tected in exosomes and microvesicles. In particular the majority of double-stranded DNA seems to be associated with tumor derived exosomes (Tha-kur BK et al. Cell research 24.6; 2014) where it represents the entire genome of the cancerous tumor from which exosomes were derived. This disco-

very corroborates the potential of exosomes, which can be easily obtained from a simple blood test. HBM is the first company to develop a kit for the isolation of circulating and Exosome-associated genomic DNA, EXO-DNA.

EXO-DNA: Isolation of circulating and Exosome-associated genomic DNA



5Exosome DNA Extraction Kits

45

EXO-DNA Isolation of circulating and Exosome-associated DNA

EXO-DNA Kit combines the ability of EXO-Prep to isolate exosomes from a wide range of biofl uids (plasma, urine, serum etc.) or culture supernatants with a user friendly system of DNA purifi cation. Isolated exosomes are lysed with the appro-priate lysis buff er and exosome DNA is purifi ed by spin columns and optimized buff ers with a fast turnaround time (approximatively 30 minutes). In addition EXO-DNA Kit provides lyophilized exosomes to be used as quality controls for exosome capture and DNA extraction.

* Pellet of exosomes aft er isolation can be treated with Dnase to eliminate cell free circulating DNA. Aft er Dnase treatment DNA extraction from microvesicle proceeds following the same protocol (see example at page 46).

Cat. Code Description Size


HBM-DNA-PS-20/# Isolation of circulating and exosome associated DNA from plasma and serum.

20 reactions

HBM-DNA-PS-40/# 40 reactions

HBM-DNA-UC-20/# Isolation of circulating and exosome associated DNA from urine and cell culture media.

20 reactions

HBM-DNA-UC-40/# 40 reactions



EXO-Prep Reagent for exosome isolation.

Lysis buff er Buff er for exosome lysis.

Washing buff ers Two washing buff ers for DNA purifi cation in spin column.

Elution Buff er Buff er for RNA elution from spin column.

Columns and Tubes Spin columns for RNA purifi cation and tubes for RNA collection.



Applications• Purifi cation of circulating

and exosome-associated DNA.

• Direct exosome capture and DNA purifi cation from biofl uids of cell media without time consuming purifi cation steps.

• Isolation of genomic exosome-associated DNA by DNAse treatment.

• Profi ling of exosome associated genomic DNA.

Advantages• Time saving procedure.

• Th e only kit on the market providing Exosome Standards as control.

• Nucleic acids extracted from a small volume amount.

• Possibility to profi le together circulating and exosome-associated genes.

5 Exosome DNA Extraction Kits

46

PerformancesExosome-associate DNA is suitable for point mutation analysis by allele-specifi c PCR.Healthy donor serum was spiked with 100 μg of purifi ed exosome from BRAFV600E-positive A375 melanoma cell lines. Vesicles were isolated by chemical precipitation with EXO-Prep contained in EXO-DNA kit and treated with or without Dn-ase 1, to distinguish circulating + Exosome related and only Exosome related DNA. Following digestion, DNA was extracted with EXO-DNA kit and analyzed by bioanalysis and allele-specifi c qPCR (Fig 1 and 2).

EXO-DNA Kit guarantees high effi ciency isolation of circulating and EV-associated DNAAmplifi cation of beta-actin from exosome-derived DNA. Exosomes were isolated from serum with our without artifi cial spike (A375-derived exosomes) using EXO-Prep solution and treated (or not) with DNAse I. DNA was extracted with HBM EXO-DNA kit and competitor and beta actin was amplifi ed by qPCR.

24

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36- DNase + DNase

Ct V

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luor

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nce)

BRAF WT

BRAF V600E

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35Serum Serum

DnaseSerum A375

Serum A375

DNAse

Ct V

alues

(Flu

ores

cenc

e)

HBM

Competitor

1. Electropherograms of genomic DNA extract-ed with EXO-DNA Kit with or without Dnase treatment

2. BRAF WT (wild type) and BRAF V600E amplifi cation by qPCR from DNA extracted with EXO-DNA Kit with or without Dnase treatment.

3. Beta-actin amplifi cation from exosome-derived DNA, extracted with HBM EXO-DNA Kit and a competitor kit for circulating DNA isolation.

Antibodies for exosome research6Summary Section 6Introduction 48Antibodies for Exosome research list 49Antibody Specifications 50

6 Antibodies for exosome research

48

IntroductionAntibodies are an essential tool for basic research, development of diagnostic tests and therapeutics for human disease. Extracellular vesicles (EVs), including exosomes, express antigens with 3D confor-mations and/or post-translational modifications that often differ from the cellular counterpart. For this

reason, most of the antibodies currently available on the market fail to recognize exosome-associated antigens with sufficient sensitivity and specificity. In collabora-tion with Exosomics Siena Spa, HBM has validated a list of polyclonal and monoclo-nal antibodies against common (CD63, CD81, ALIX) and disease- specific (cancer and neurodegenerative diseases) exosomal markers.



6Antibodies for exosome research

49

Antibodies for Exosome ResearchCat. Code Antibody Type Applicartions

Monoclonal Antibodies

HBM-CD9-### Anti human CD9 Mouse monoclonal unconjugated WB, FACS, ELISA, IP, IHC

HBM-CD9B-### Anti human CD9 Mouse monoclonal biotin conjugated WB,, ELISA, IP

HBM-CD63-### Anti human CD63 Mouse monoclonal unconjugated WB, FACS, ELISA, IP, IHC

HBM-CD41-EM1-### Anti human CD41 Mouse monoclonal unconjugated FACS, ELISA

HBM-CD81-EM4-### Anti human CD81 Mouse monoclonal unconjugated WB, FACS, ELISA, IP, IHC

HBM-CD44-EM1-### Anti human CD44 Mouse monoclonal unconjugated WB, ELISA, IP

HBM-CAV1-D4-### Anti human Caveolin 1 Mouse monoclonal unconjugated WB, FACS

HBM-ALIX-### Anti human Alix Mouse monoclonal unconjugated WB, FACS, IF

HBM-SF4-### Anti human TM9SF4 Mouse monoclonal unconjugated WB, FACS, IP

HBM-CD9M-### Anti mouse CD9 Mouse monoclonal unconjugated WB, FACS, ELISA, IP

Polyclonal Antibodies

HBM-FLOT-### Anti human Flotillin Rabbit polyclonal unconjugated

WB,FACS, ELISA

HBM-RAB5-PR1-### Anti human RAB5 Rabbit polyclonal unconjugated

WB, FACS, ELISA, IP

HBM-HSP70-SR1-### Anti human HSP70 Rabbit polyclonal unconjugated

WB, FACS, ELISA

HBM-SF4-PR2-### Anti human TM9SF4 Rabbit polyclonal unconjugated

WB, IP


WB, FACS, IP


WB, ELISA, IHC

HBM-TSTA3-PR1-### Anti human TSTA3 Rabbit polyclonal unconjugated

WB, ELISA

Abreviation list:

ELISA: Enzyme-Linked Immunosorbent AssayFACS: Fluorescent-Activated Cell SortingIHC: ImmunohistochemistryIP: Immuno precipitationIF: ImmunofluorescenceWB: Western Blotting

Applications• Exosome marker detection

in different techniques.

• Exosome immunocapture.

Advantages• All HBM antibodies are

tested and validated for exosome antigen reactivity.

• Available in 2 sizes: 50 μg and 100 μg.

Shipment and storage• All HBM antibodies are

shipped at 4°C. Storage conditions are described in Antibody datasheet.

6 Antibodies for Exosome Research

50

Anti human CD9 antibodyDescription Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24)

belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abun-dant on exosome membranes. HBM off ers monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specifi c antigen on both cell lines and extracellular vesicles.

Cat Num/Amount HBM-CD9-050 (50 μg)HBM-CD9-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHCReferences - Rampini, S., et al. “Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and

single cells.” Lab on a Chip 15.16 (2015): 3370-3379.- Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-form.” Analytica Chimica Acta (2015).

Anti human CD9 antibody biotin conjugatedDescription Anti-CD9 recognizes an human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24)

belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM off ers two monoclonal anti-CD9 antibodies, unconjugated or biotin conjugated, recognizing the specifi c antigen on both cell lines and extracellular vesicles. HBM antibodies are compatible with ELISA, WB and fl ow cytometry applications.

Cat Num/Amount HBM-CD9B-050 (50 μg)HBM-CD9B-100 (100 μg)

Type Mouse monoclonal biotin conjugatedReactivity HumanApplications WB, ELISA, IP

Western Blott ing1- MM1 (melanoma cell) lysate (20 µg)2- MM1 cells purifi ed exosomes (20 µg)3-Plasma healthy donors purifi ed exosomes (20 µg)

FACS CD9 staining of human plasma purifi ed exosomes

ELISACD9 detection in purifi ed exosomes from human plasma (PEP) and urine (PEU), 30 µg

Western blott ing1- COLO1 cell lysates (20 µg)2- CD9 detection in plasma purifi ed exosomes (20 µg)

ELISACD9 detection in purifi ed exosomes from hu-man plasma (PEP) and urine (PEU), 30 µg

6Antibodies for Exosome Research

51

Anti human CD63 antibodyDescription Anti-CD63 recognizes an extracellular fragment of CD63, a 56 kiloDalton (kDa), type III lysosomal

glycoprotein, belonging to the tetraspanin family. CD63 is expressed by granulocytes, platelets, T-cells, monocytes/macrophages and endothelial cells. CD63 protein is a canonical exosome marker and currently used to characterize exosome populations from a variety of body fl uids.

Cat Num/Amount HBM-CD63-050 (50 μg)HBM-CD63-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHC

Anti human CD41 antibody Description Integrin alpha chain 2b, also known as CD41, is an heterodimeric integral membrane protein. It

undergoes post-translational modifi cations that result in two polypeptide chains linked by a di-sulfi de bond. CD41 is expressed on platelets and megakaryocytes, but also on early embryonic hematopoietic stem cells and related exosomes.

Cat Num/Amount HBM-CD41-EM1-050 (50 μg)HBM-CD41-EM1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications FACS, ELISA

ELISA CD63 detection in purifi ed exosomes derived from cell supernatants (COLO1-30, MM1-30, BLCL21-30)

FACS CD63 staining of human plasma purifi ed exosomes

FACSStaining of CD41in human plasma puri-fi ed exosomes (PEP-30)

ELISAPEP-30: 30 ug of purifi ed exosomes from human healthy donors plasma.MM1-30: 30 ug of purifi ed exosomes from MM1 cell supernatantCOLO1-30: : 30 ug of purifi ed exosomes from COLO1 cell supernatant


52

Anti human CD81 antibodyDescription CD81 (TAPA1), a member of the tetraspanin family, is virtually expressed on all nucleated cells, but

in particular on germinal center B cells. CD81 forms complexes with other tetraspanin proteins, integrins and coreceptors. CD81 is expressed on exosome membranes. HBM anti-CD81 mono-clonal antibody is adapt for specifi c antigen recognition from cell lysates and exosomes using the techniques indicated below.


Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IP, IHCReferences - Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-

form.” Analytica Chimica Acta (2015).

Anti human CD44 antibody Description CD44, known also as HCAM, is a 742 aminoacids protein involved in lymphocytes activation, hom-

ing and hematopoiesis, CD44 is expressed in multiple isoforms. CD44 is highly expressed in cancer tissues and tumoral-derived exosomes, suggesting a role in tumor progression and metastasis.


Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, ELISA, IP

Western blott ing1- MM1 (melanoma cell) lysate (20 ug)2- MM1 cells purifi ed exosomes (20 ug)3-Plasma healthy donors purifi ed exo-somes (20 ug)

FACS CD81 staining of COLO1 cell purifi ed exosomes

ELISACD81 on purifi ed exosomes from urine (PEU), 30 ug

Western blott ing1- 20ug of whole lysate of MM1 cell super-natant purifi ed exosomes 2- 20 ug of purifi ed exosomes from human plasma of healthy donors (PEP)

ELISAPEP-30: 30 ug of purifi ed exosomes from human healthy donors plasma.MM1-30: 30 ug of purifi ed exosomes from MM1 cell supernatantCOLO1-30: : 30 ug of purifi ed exosomes from COLO1 cell supernatant


53

Anti human Caveolin antibodyDescription Th e lipid raft -associated protein caveolin-1 (CAV1) is the major component of the inner surface

of caveolae, small invaginations of the plasma membrane. Caveolin is a transmembrane adaptor molecule that can simultaneously recognize GPI-linked proteins and interact with downstream cytoplasmic signaling molecules. It is highly expressed on exosomes derived from tumor tissue.

Cat Num/Amount HBM-CAV1-D4-050 (50 μg)HBM-CAV1-D4-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS

Anti human Alix antibody Description Alix protein, named also ALG2 interacting protein or PDCD6-interacting protein, is a cytoplas-

mic protein that interacts with apoptosis associated-proteins. Alix plays an active role in exosome biogenesis and it is a useful internal marker for the analysis of exosomal proteins with western blott ing.

Cat Num/Amount HBM-ALIX-SM1-050 (50 μg)HBM-ALIX-SM1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS, IF

FACSStaining of Cav1 in MM1 purifi ed exosomes

Western blott ing 1-20 ug of whole lysate of melanoma cell derived exosomes (MM1) 2- 20 ug of whole MM1 cell lysate

Immunofl uorescenceImmunofl uorescence staining of HeLA cells.

Western blott ing1- MM1 (melanoma cell) lysate (20 ug)2- MM1 cells purifi ed exosomes (20 ug)3-Plasma healthy donors purifi ed exosomes (20 ug)


54

HBM-MM1 (Melanoma cell

Exosome Standards)

HBM-PEP(Plasma healthy donor Exosome Standards)

Western blott ing1- 20 ug of whole lysate of melanoma cell line2- 20ug of whole lysate of MM1 cell superna-tant purifi ed exosomes 3- 20 ug of purifi ed exosomes from human plasma of healthy donors (PEP)

FACSStaining of TM9SF4 on MM1 purifi ed exosomes vs human plasma exosomes (PEP) demonstrates the predominant TM9SF4 expression in exosomes derived from tumoral source

FACS CD9 staining of B16F10 mouse cell pu-rifi ed exosomes

Anti human TM9SF4 antibodyDescription TM9SF4 (TUCAP1) is a newly discovered tumor-associated protein of unknown function that

belongs to the Trans-Membrane 9 Superfamily (TM9SF). Th ese proteins are characterized by the presence of a large variable extracellular N-terminal domain followed by nine putative transmem-brane domains and a conserved C-terminal domain. TM9SF4 is mainly expressed on exosomes derived from tumor tissue. HBM off ers three diff erent monoclonal antibodies that recognize the protein on both exosomes and cell lysates.

Cat Num/Amount HBM-SF4-PMG1-050 (50 μg)HBM-SF4-PMG1-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity HumanApplications WB, FACS, IP

Anti mouse CD9 antibodyDescription Anti-CD9 recognizes a human 24-kiloDalton (kDa) single-chain cell-surface glycoprotein (p24)

belonging to the tetraspanin family. CD9 has a very broad tissue distribution and is abundant on exosome membranes. HBM off ers a monoclonal anti-CD9 antibody unconjugated reactive against mouse antigen and able to identify CD9 in mouse cell of biofl uids derived exosomes.

Cat Num/Amount HBM-CD9M-050 (50 μg)HBM-CD9M-100 (100 μg)

Type Mouse monoclonal unconjugatedReactivity MouseApplications WB, ELISA, FACS, IP, IHC

1 2

Western blott ing1- 20ug of whole lysate of B16F10 cell super-natant purifi ed exosomes 2- 20 ug of exosomes isolated from mouse plasma


55

Anti human Flotillin antibodyDescription Flotillin belongs to the band 7.2/stomatin protein family and appears to be strongly expressed in

muscle cells and fi broblasts. Flotillin expression is also correlated with Alzheimer development. Flo-tillin is highly expressed on exosomes and appears to be involved in exosome release mechanism. It is considered a common marker for exosomes analyses.

Cat Num/Amount HBM-FLOT-SR1-050 (50 μg)HBM-FLOT-SR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACSReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


Anti human Rab5 antibodyDescription Rab5 is a small GTPase belonging to Ras superfamily of monomeric G proteins. Rab GTPases play

an essential role in the regulation of membrane traffi c and are involved in vesicle formation and trasport and fusion to the membrane. Rab5 is expressed on exosome membranes and it might have an active role during endo/exocytotic processes of microvesiscles through the plasma membrane.

Cat Num/Amount HBM-RAB5-PR1-050 (50 μg)HBM-RAB5-PR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IPReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


FACS Purifi ed exosomes from MM1 (30 ug)

stained by anti-Flotillin antibody

ELISA Detection of diff erent exosomes purifi ed from cell culture supernatants or human plasma (HD), performed with anti-Flotillin

Western blott ingExosomes purifi ed from 0.5, 0.25, 0.1 ml of hu-man plasma demonstrate diff erent intensity of Rab5 expression

0.5 ml 0,25 ml 0,1 ml

FACS Purifi ed exosomes from COLO1 cell line detected by Rab5 anti-body


56

Anti human HSP70 antibodyDescription Heat shock protein 70 (HSP70) is a molecular chaperone that facilitates the assembly of multi-

protein complexes and traffi cking of polypeptides across cell membranes. HSP70 is active in pro-moting tumorigenesis and functions as an anti-apoptotic factor. It is highly expressed on exosomes derived from tumor tissues.

Cat Num/Amount HBM-HSP70-SR1-050 (50 μg)HBM-HSP70-SR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA, FACS, IPReferences Gagni, Paola, et al. “Combined mass quantitation and phenotyping of intact extracellular vesicles by a microarray plat-


Anti human TM9SF3 antibodyDescription TM9SF3 belongs to nonaspanins family proteins (TM9SF), characterized by a large non-cyto-

plasmic domain and nine putative transmembrane domains. Although litt le is known about the functions of this protein, it might participate in tumor invasion and it is emerging as a potential prognostic factor. Protein is expressed on plasma membranes and exosomes.

Cat Num/Amount HBM-SF3-PR1-050 (50 μg)HBM-SF3-PR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA,IHC

Western blott ingAnalysis of HSP70 expression in diff erent exosomes derived from tumoral cell lines:1- COLO1; 2- SH-SYSY; 3 - U87; 4 - SK

FACS Staining of COLO1 derived exosomes with anti-HSP70

ElisaTM9SF3 detected in diff erent purifi ed exosomes im-munocaptured on EXOtest plate

Western blott ingDetection of TM9SF3 in 1 - MM1 cell lines (20 µg)2 - MM1 derived exosomes (20 µg)


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Anti human TM9SF4 antibody Description TM9SF4 (TUCAP1) is a newly discovered tumor associated protein that belongs to the Trans-

Membrane 9 Superfamily (TM9SF). Th ese proteins are characterized by the presence of a large variable extracellular N-terminal domain followed by nine putative transmembrane domains and a conserved C- terminal domain. TM9SF4 is mainly expressed on exosomes derived from tumor tis-sue. HBM off ers two rabbit polyclonal antibodies that recognize the protein from both exosomes and cell lysates.

Cat Num/Amount HBM-SF4-PR2-050 (50 μg)HBM-SF4-PR2-100 (100 μg)HBM-SF4-PR3-050 (50 μg)HBM-SF4-PR3-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications HBM-SF4-PR2: IP, FACS, IHC

HBM-SF4-PR3: WB, IP

Anti human TSTA3 antibodyDescription Tissue specifi c transplantation antigen P35B is a NADP(H)-binding protein. It catalyzes the two-

step epimerase and the reductase reactions in GDP-D-mannose metabolism, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. TSTA3 have been identifi ed in the overall exosome population.

Cat Num/Amount HBM-TSTA3-PR1-050 (50 μg)HBM-TSTA3-PR1-100 (100 μg)

Type Rabbit polyclonal unconjugatedReactivity HumanApplications WB, ELISA

Western blott ingHBM-SF4-PR220 ug of whole HEK293 cell lysate (1), 20 ug of exosomes purifi ed from MM1 cell supernatant (2) and from human plasma of healthy donors (3)

FACS Purifi ed exosomes from MM1 cell line detected by HBM-SF4-PR1 antibody

IHCImmunohystochemical staining of TM9SF4 in specimens derived from healthy colon.

Western blott ingDetection of TSTA3 in:20 ug COLO1 purifi ed exosmes. 20 ug of exosomes purifi ed human plasma (PEP)*in purifi ed exosomes from plasma TSTA3 can ap-pear in dimer form (signal around 70 kDa)


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Summary Section 7Introduction 60Exosome and Microvesicle purifi cation and analysis 61

RNA and DNA profi ling and Next-Generation-Sequencing 61Custom products 62Additional services and project collaboration

Custom Services and Collaborations7

7 Custom Services and Collaborations

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Introduction

Being the oldest company entirely dedicated to exosome research, since 2007 HansaBioMed has developed unique competences and tools allowing us to help you with your research work. Our experienced scientists will help you accelerate your research and achieve your objectives quickly, providing consultancy and/or professional services using the state of art equipment. Our services include:

1- Exosome and Microvesicle purifi cation from diff erent biological samples and complex matrices.

2- Exosome RNA and DNA profi ling and Next Generation Sequencing.

3- Custom products (immunoplates for exosome capture, immunobeads, ExoTESTTMkits).

4- Modifi cation of biomolecules.

5- Development of new tools for capture and enrichment of exosome subpopulations or quantifi cation of specifi c markers.

6- Project collaborations and technical consulting.


7Custom Service and Collaborations

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Exosome and Microvesicle purification and analysis

Our Exosome Custom Service offers purification of Exosomes and Microvesicles from a wide range of biological samples (human and animal) and cell culture me-dia. Exosome purification can be obtained using different methods according to your sample characteristics and your requests (combination of ultracentrifugation and microfiltration steps, immunocapture, chemical precipitation). Exosome can be subsequently quantified for total protein content and validated by NTA (NanoSight LM10). Purified exosomes can be shipped to the customer lyophilized or frozen, as required. Moreover, we offer additional services to facilitate your research such as those listed below.

Service Description

Exosome and Microvesicle Isolation Exosome and Microvesicle isolation via ultracentrifuge or other methods.

Isolation of exosome miRNA Extraction of miRNA from exosomes. miRNA will be shipped to the customer in dry ice.

Exosome protein marker profiling Profiling of exosome protein marker via quantitative ELISA, FACS analysis or Mass Spectrometry.

RNA and DNA profiling and Next-Gen- Sequencing (NGS)

Hansabiomed scientists can provide an expert service for isolation and identification of exosome-associated RNA/DNA biomarkers leveraging the throughput and scal-ability of Ion Torrent and SOLiD System (Thermo Fisher Scientific) next-generation sequencing platforms. This service is an easy solution for researchers who are inter-ested in identifying new exosome RNA biomarkers or in the understanding the pro-filing expression of such biomarkers in the exosomes of their model cellular systems or patient biofluids. You will receive your data back in approximately 4-6 weeks.

Service Description

RNA and DNA Next Gen Sequencing Extraction of RNAs and DNA vesicle associated and NGS service.

Isolation of exosome RNAs or genomic DNA and profiling

Extraction of RNAs (miRNA and/or total RNA) and genomic DNA from exosomes. Profiling of nucleic acid marker by qRT-PCR. Alternatively RNA or DNA can be shipped to the customer in dry ice.

Advantages• Exosome purification and

services performed by experienced scientists and qualified personnel.

• Material is processed quickly.

• HansaBioMed has all the necessary facilities for exosome purification, protein and nucleic acid profiling.

7 Custom Services and Collaborations

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Custom productsHansaBioMed can design and create a personalized kit by choosing from among a wide variety of reagents and tools in our catalog.

Custom product Description

Custom ELISA immunoplate

1- Choose your preferred plate (trasparent, white or black) depending on your final application.2- Choose the antibody from our antibody list (see section 6) or send us the antibody for plate coating.

Custom ExoTESTTM Ready to use kits

1- Select the plate/beads that provides the capture or enrichment of exosome population of interest.2- Select the Exosome Standards you need.3- Select the primary antibody you want to use for exosome detection.

Custom immunobeads Latex immunobeads will be covalently coupled with the antibody of your interest, chosen from our antibody list.

For other Custom made solutions and for informations contact us at: [email protected]

Additional services and project collaboration

HansaBioMed is open for collaboration with private or academic partners in re-search activites related to different fields of exosome research and applications. Contact us for:

Service Description

Biomolecule labeling and modification

We can provide biomolecule modifications such as: protein labeling with fluorescence dyes, conjugation of biomolecules with affinity ligands, PEGyation of biomolecules, immobilization of biomolecules on dif-ferent surfaces (hydrogels, molded polymers, synthetic membranes) etc.

Development of new assays for exosome marker identification

We can develop personalized ELISA assays for a specific exosome protein marker quantification or qPCR assays for identification of miRNA and DNA markers.

Development of tools for capture and enrichment of specific exosome subpopu-lations

We can develop tools for capture and isoaltion of specific exosome subpopulations to improve exosome comprehensive profiling in diseases.

Project and collaborations and technical consulting

Contact us for: OEM product development, cell free re-generative medicine, collaboration in research projects (Horizon 2020, Eurostars, NIH grants, etc).

Cell Fractionation Kits

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Cell

Cell Organells

Nuclei

Cytoskelatal fraction

Cytosol fraction

Membrane fraction

Nuclear fraction

Cytosol extraction

Membrane extraction

Nuclear extraction

Product code Product name Description

HBM-K270-50 Fraction-PREP Cell Fractionation Kit

Th e Fraction-PREPTM Cell Fractionation System is designed to fractionate cellular proteins into four distinct protein fractions: Cytosolic fraction, mem-brane fraction, nuclear fraction, and cytoskeletal fraction. All four protein fractions can be directly used in downstream assays, such as 2-D gel, Western blot, gel-shift assay, translocation, enzyme activity assays, and reporter assays, etc.

HBM-K256-100 Mitochondria/Cytosol Fractionation Kit Separate highly enriched mitochondria from cytosol of mammalian cells. Simple procedure with no ultracentrifugations or toxic chemicals involved.

HBM-K266-100 Nuclear/Cytosol Fractionation KitSeparate nucleus from cytoplasm in less than 90 minutes. Th e kit provides a system that maintains the nuclear and cytosol compartments separated. Th e separated fractions are suitable for downstream assays.

HBM-K268-50 Membrane Protein Extraction KitIsolate integral membrane and/or plasma membrane proteins effi ciently from cultured mammalian cells. Th e isolated membrane proteins can directly be used in downstream assays.

HBM-K269-500 Mammalian Cell Extraction KitExtract mammalian proteins from tissues and cells under nondenaturing conditions. Cell extracts can be used in a variety of assays, such as enzyme activity assays, Western blot, and others.

HBM-K280-50 Mitochondrial DNA Isolation KitTh e kit provides unique formulations of buff ers and reagents for isolating a highly enriched mitochondrial fraction. Mitochondrial DNA are then isolated using reagents included in the kit

HBM-K904-### PEG virus Precipitation Kit

HansaBioMed’s PEG Virus Precipitation Kit provides an easy, convenient and time-saving method to concentrate virus without ultra-centrifugation. Th e kit is able to concentrate retroviruses, baculoviruses, lentiviruses, and phages etc. in cell culture medium or environmental samples.

Separe diff erent cell compartments with HansaBioMed Cell Fractionation Kits.

HansaBioMed off ers a comprehensive selection of systems for convenient fractionation of diff erent cellular compartments. Th ese kits provide unique formulations of buff ers and reagents for conve-nient preparation of highly enriched cellular frac-tions from mammalian cells and tissues. Th e prepa-red protein fractions are suitable for downstream assays, such as two-dimentional gel, protein immu-noblott ing, gel-shift assay, enzyme activity assays, reporter assays, and more.

Cell Proliferation and Viability Kits

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Easy and fast analysis of cell viability and cell proli-feration assays.Proliferation or viability analyses are crucial for cell growth and diff erentiation studies, and are oft en coupled with metabolism analysis. Metabolic activity is commonly used as a viability indicator, but for some applications it can be important to assess viability independent of metabolic state and this is where the cell viability assays come in play. Proliferation is also a convenient measure of population dynamics in studies of cytokines or growth factors, or in bioprocess optimization work. HansaBioMed off ers a pa-nel of diff erent solutions for a fast and easy determination of the cell viability and proliferation in your cell cultures.

Product code Product name Description

HBM-K306-### BrdU Cell Proliferation Assay KitCompared to other cell proliferation assays, this kit detects only the prolife-rating cells and not the seeded cells. Th is highly sensitive, non-radioactive kit detects as less as 50-100 proliferating cells.

HBM-K501-100 Live-Dead Cell Staining Kit

Th e Live-Dead Cell Staining Kit provides the ready-to-use reagents for convenient discrimination between live and dead cells. Stained live and dead cells can be visualized by fl uorescence microscopy using a band-pass fi lter (detects FITC and rhodamine)

HBM-K300-### MTS Cell Proliferation Colorimetric Assay Kit

MTS Cell Proliferation Assay Kit is a colorimetric method for sensitive quan-tifi cation of viable cells in proliferation and cytotoxicity assay. . It can also be used for the analysis of cytotoxic compounds like anticancer drugs and many other toxic agents and pharmaceutical compounds

HBM-K245-100 Proteasome Activity Assay Kit

Proteasome Activity Assay takes advantage of the chymotrypsin-like activity, utilizing an AMC-tagged peptide substrate which releases free, highly fl uo-rescent AMC in the presence of proteolytic activity. it allows diff erentiation of proteasome activity from other protease activity which may be present in samples

HBM-K302-### Quick Cell Proliferation Assay Kit

Th e assay is based on the cleavage of the tetrazolium salt to formazan by cellular mitochondrial dehydrogenase. Th e amount of the dye generated by activity of dehydrogenase is directly proportional to the number of living cells.

HBM-K320-50 Senescence Detection KitTh e Senescence detection kit is designed to histochemically detect SA-β-Gal activity in cultured cells and tissue sections, a known characteristic of senescent cells.

HBM-K771-100 Trypsin Activity Assay KitTh e kit detects 10-100 mU (p-NA unit) trypsin in various samples. . In the assay, trypsin cleaves a substrate to generate p-nitroaniline (p-NA) which is detected at λ=405 nm.

HBM-K304-2500 WST-1 Cell Proliferation Reagent

Th e ready-to-use cell proliferation reagent, WST-1 provides a simple and accurate method to measure cell proliferation, which is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases.

HansaBioMed LLCAkadeemia tee 15, 12618 Tallinn, ESTONIAwww.hansabiomed.eu

Email: [email protected]: +372 6204348

Contact in USGalen Laboratory SuppliesP.O. Box 682Middletown, CT 06457www.galenlabsupplies.com

Email: [email protected]: 860-704-9058Fax: 888-260-6091

Contact in Russia and CIS countriesAlgimed LTDLogoyskiy Tract 22/1-35B220090 Minsk, BELARUSwww.algimed.by

Email: [email protected]: +375 172 024 302Fax: +375 172 024 338

Documents

Optimized solutions catalog.pdf · Exosomes are subsequently quantifi ed and validated for overall protein content and ... (Human being prostatic ... profi le of common exosomal markers