Cellutions 2011V2 - The Newsletter for Cell Biology Researchers

8/6/2019 Cellutions 2011V2 - The Newsletter for Cell Biology Researchers

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EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

Cellutions

Vol 2: 2011The Newsletter forCell Biology Researchers

ChangeCell Fate

Fluorescent GelatinDegradation Assays forInvestigating InvadopodiaFormation Page 8

Exploring Akt/mTOR SignalingUsing the MILLIPLEXmap Akt/mTOR 11-plex PanelPage 13

Immuno-monitoring Using theScepter 2.0 Cell Counterand Software ModulePage 16

Mitochondrial to Nuclear DNARatio: Sensitive Biomarkerof Stem Cell DifferentiationPage 23

Exciting New Products forCell Biology ResearchPage 27

To subscribe to the quarterly Cellutions newsletter,please visit www.millipore.com/cellquarterlynews

Enhanced Reprogramming of Human SomaticCells Using Human STEMCCA PolycistronicLentivirus and Human iPS Cell BoostSupplement page 3
http://www.millipore.com/reply/form/cellquarterlynews?open&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/reply/form/cellquarterlynews?open&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/reply/form/cellquarterlynews?open&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RC


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Visitwww.millipore.com/OPCdiffkit to learn more.

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Successful in vitro myelination using the oligodendrocyte

differentiation kit. After 21 days of coculture with Human OPCs,the axons of rat primary neurons (green) are encased by MBP-positive (cyan) cells.
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AbstractThe ability to reprogram differentiated adult cellsto a state that resembles embryonic stem cells hascreated wide-ranging opportunities for development of relevant in vitro disease models and patient-speci c cellreplenishment therapies.

Initial efforts to generate human induced pluripotentstem cells (iPS cells) required simultaneous co-infectionof cells with four separate retroviral expression vectors(Oct-4, Klf4, Sox-2 and c-Myc). Each vector carried onetranscription factor, which resulted in a high number of

genomic integrations. Single polycistronic lentiviral vectors,such as those in EMD Millipores STEMCCA lentivirusreprogramming kits, can improve ef ciency and reducethe number of viral integrations. In one report, highpercentages of disease-speci c human iPS clones wereisolated which possessed only a single viral integrant2.Despite these advances, reprogramming human somaticcells remains a highly inef cient and time-consumingprocess. Small molecules targeting speci c signalingpathways have been shown to enhance reprogrammingand/or replace the transcription factors required forreprogramming5-7. Here, a cocktail of small moleculeswas identi ed that, when used in conjunction with theSTEMCCA kits, further increased reprogramming ef ciency,decreased time required to establish full reprogramming,and maintained the desired iPS cell morphology andpluripotency.

IntroductionWe have previously developed mouse STEMCCA lentiviruskits that have been validated for the generation of both

mouse and human iPS cells from mouse embryonic

broblasts (MEFs) and human foreskin broblasts (HFFs),respectively. However, a high multiplicity of infection (MOI

= 200) was required for reprogramming human somaticcells. The recently launched human STEMCCA kits replacedmouse genes with human transcription factors, yieldingsimilar numbers of colonies but at a dramatically reducedMOI (MOI = 20). Both human and mouse STEMCCAlentivirus kits are available in constitutive and Cre/LoxP-regulated formats for higher reprogramming ef ciency of normal and diseased post-natal somatic cells2-4.

Although lenti- and retro-virus based reprogrammingremain the most ef cient methods to deliver exogenousreprogramming factors into the host cell genome, humaniPS cell generation is still slow (around 4 weeks) andinef cient (0.01-0.1% ef ciency), resulting in mixedpopulations of partial and full reprogrammed colonies.Substantial effort and progress have been made togenerate iPSCs with fewer or no exogenous geneticmanipulations for example, by introduction of chemicalcompounds that can functionally replace reprogrammingtranscription factors and/or enhance ef ciency and kineticsof reprogramming5-7. Human keratinocytes8 and mouse

broblasts9 have now been reprogrammed to iPSCs with asingle gene, Oct4, and a cocktail of small molecules.

Here, various combinations of small molecules involvedin TGFa , Wnt, and MAPK signaling pathways along withepigenetic modi ers were screened for their effectson (1) increasing the ratio of fully reprogrammedSSEA4+TRA-1-60+ Hoechstdim iPS cells versusreprogramming intermediates; (2) increasing colonynumbers and (3) reducing the time to establishment of fullreprogrammed iPS cell colonies.

Enhanced Reprogramming of Human Somatic Cells usingHuman STEMCCA PolycistronicLentivirus and Human iPS CellBoost SupplementMin Lu, Cristina Moore, Vi ChuEMD Millipore


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From this screen, a small molecule boost supplementwas identi ed which in combination with the STEMCCAlentivirus kits ful lled the three aforementioned criteria.The small molecule boost supplement was validated onfeeder-based (KOSR) and serum-free, feeder-free culture

systems and was furthermore shown to be highly effectivein reprogramming different primary broblast cell lines.

Materials and MethodsThe reprogramming protocol followed is outlined in Figure1. FibroGRO Xeno-free Human Foreskin Fibroblasts(HFFs, Cat No. SCC058) were seeded at a density of 10,000cells/well and transduced with the Human STEMCCAConstitutive Polycistronic Lentivirus Reprogramming Kit(Cat No. SCR544). Cells were replated onto irradiated MEFsin the presence or absence of a panel of small moleculesat day 6, using the human ESC medium of choice (eitherKOSR-based media on inactive MEFs (Cat No. PMEF-CF) oron Matrigel-coated or Geltrex- coated plates if usingmTeSR or StemPRO media, respectively).

The medium was changed every other day and colonieswere picked when they reached several hundred cellsin size. A total of 25 small molecule boost cocktails(proprietary formulations) were tested and several

treatments were able to give rise to large at 2D coloniesthat displayed morphological features similar to hESCs andcould be easily passaged.

Colonies were stained with anti-Tra-1-60 (Cat. No.

FCMAB115F)and anti-SSEA-4(Cat. No. MAB4304). Tra-1-60 is a marker that is expressed at a later stage of reprogramming10. Tra-1-60 and SSEA-4 double positivestaining has been suggested to be a valid marker for fullreprogrammed iPSCs11.

esultsHFF were transduced with STEMCCA lentivirus, replatedonto irradiated MEFs in the presence or absence of a panelof small molecules at day 6, and evaluated for number of colonies generated, morphology, ease of passaging, anddays to passage (Figure 2, 3). Chemically treated humaniPSCs possessed fast proliferation kinetics; early passagesfrom P0 to P3 were shortened to 5-6 days per passageperiod - a timeframe that is similar to the proliferationrate of normal hESC (Figure 3). Treatment 2 was selectedon the basis that it signi cantly improved both the qualityof colonies formed and the ef ciency of reprogramming.Treatment 2 is herein referred to as Human iPS Cell Boost.

Figure 1. Time courseschematic of reprogramminghuman somatic cells usingSTEMCCA polycistroniclentivirus kits combinedwith Human iPS Cell BoostSupplement.

Figure 2. Small molecule combinations were screened for their effects on colony morphology, number of colonies generated, easeof passaging and relative proliferation rate as measured by days to passaging. Treatment 2 was selected for further characterization.Treatment 2 is referred to as Human iPS Cell Boost Supplement.

ntreated reatment 1 reatment 2 reatment 3

Morphology: 3D Flat 2D Flat 2D Flat 2D

# Colonies: 1x 3.6x 2.6x 0.5x

Passageability: Dif cult Easy Easy Easy

Days to Passage: 10-12 days 5-6 days 5-6 days 5-6 days

Day

Seed HFFs or target cells

Infect with virus

Infect with virus

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 9 20 21 22 23 24 25

Medium

Prepare feeders

Replate on feeders

Prepare feeders

Pick colonies

Add feeders to culture

FibroGRO LS Complete Medium

Human iPS Cell Boost Supplement

Human ESC Media

MEFMEF
http://www.millipore.com/catalogue/item/FCMAB115F&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/MAB4304&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/MAB4304&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/FCMAB115F&cid=BIOS-S-EPDF-1189-1108-RC


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In the presence of the Human iPS Cell Boost Supplement,the number of colonies formed increased 3-fold (Figure4A) when used in combination with the mouse STEMCCAlentivirus kits(Cat. Nos. SCR530,SCR531) and 15-foldwhen used in combination with the human STEMCCAlentivirus kits (Cat. Nos.SCR544, SCR545). Colonies stainedpositive for both human ESC markers, SSEA-4 (Figure 4F)and TRA-1-60 (Figure 4G). TRA-1-60+ colonies were not

observed in the untreated control cultures (Figure 4D).

Human iPS colonies generated in the presence of HumaniPS Cell Boost Supplement could be readily expandedfor multiple passages (over 30 passages). Human iPScells displayed the morphology characteristic of humanESCs, had a normal karyotype and stained positive forpluripotent markers (Figure 5).

Figure 4. Addition of Human iPS Cell Boost Supplement to a polycistronic lentivirus-based reprogramming regime (STEMCCA) dramatically increased the ef ciencyof colony formation (A) and shortened the time to establishment of full reprogrammed human iPS clones (E, F, G). Human iPS Cell Boost Supplement enhanced colonyformation by 2-3 fold when used in combination with the mouse STEMCCA lentivirus kits (SCR530, SCR531)and 15-fold when used in combination with the humanSTEMCCA lentivirus kits (SCR544, SCR545)(A). Four independent experiments (MOI = 200) were performed using mouse STEMCCA lentivirus kit while two experimentswere performed (MOI = 10) using human STEMCCA lentivirus kits. Reprogramming without chemical treatment was used as a control for all experiments. p0 human iPScolonies generated from FibroGRO Xeno-free Human Foreskin Fibroblasts (Cat. No. SCC058)reprogrammed with mouse STEMCCA lentivirus (SCR530)in the presence of the Human iPS Cell Boost Supplement exhibited larger colony sizes, a at 2D morphology (E), and are SSEA-4- positive (F) and TRA-1-60-positive (G). This is in markedcontrast to untreated control where the colonies are smaller, are 3D in morphology (B) and are SSEA-4 positive (C) but TRA-1-60-negative (D) at similar timepoints.

Figure 3. Proliferation kinetics similar to human ESC were obtained for all three chemicaltreatments starting at p1 (5-7 days till passaging) versus from p3 for untreated control (data notshown).

Figure 5. Human iPS cells generated using the Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Kit(SCR545) in combination with Human iPS Cell Boost Supplement possessed an apparently normal karyotype (A) and expressed theappropriate human pluripotent markers, alkaline phosphatase (B), SSEA-4 (C), Oct-4 (D), SSEA-3 (E), TRA-1-60 (F), and TRA-1-81 (G).Cytogenic analysis was performed on twenty G-banded metaphase cells from p9 human iPS cells. All twenty cells demonstrated anapparently normal male karyotype. No abnormal cells were detected (A, Cell Line Genetics).

reatment 1 reatment 2 reatment 3

P 1 (

5 -

6 )

P

2 ( 5

- 6 )

BA C D

E F G

A DB

E

C

F G

25

20

15

10

5 N o .

o f c o

l o n

i e s

Mouse STEMCCA

0Cell Boost Treated Control

60

70

50

40

30

20

10 N o .

o f c o

l o n

i e s

Human STEMCCA

http://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR531&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR531&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCC058&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCC058&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR531&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR531&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR530&cid=BIOS-S-EPDF-1189-1108-RC


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The versatility of the Human iPS Cell Boost Supplementwas further demonstrated by enhanced reprogrammingof multiple primary human broblast cell lines includingHFF and BJ (ATCC) (Figure 7) in either KOSR feeder-basedor serum-free, feeder-free culture systems (mTeSR andStemPRO) (Figure 6).

Figure 7. Day 12 human iPS colonies generated from BJneonatal foreskin broblasts using mouse STEMCCA lentivirus kitin combination with Human iPS Cell Boost Supplement exhibiteda 10-fold increase in colony numbers (E) and displayed increasedcolony size (B) relative to untreated control (A). Human iPScolonies picked at day 21 and passaged on MEFs exhibitedsimilar at 2D morphology (C, D) and similar proliferationkinetics as human ES cells (data not shown).

Figure 6. Human iPS Cell Boost Supplement improvesreprogramming ef ciency in serum-free, feeder-freebased culture systems and increased colony size relativeto untreated controls (compare B, D to A, C). HumaniPS colonies were generated using mouse STEMCCAlentivirus kit in the absence or presence of the HumaniPS Cell Boost Supplement. Cells were cultured oneither Geltrex-coated plates in StemPRO medium (A,B) or Matrigel-coated plates in mTeSR medium (C, D).Day 23 fully reprogrammed hiPS colonies were isolatedand continually passaged in mTeSR conditions exhibitedtypical hESC morphology (F).

Feeder/serumfree culture

No. of cells/well re-platedin feeder-free culture

Chemicalreatment Colony#

mTeSR 5 x 104 + 70

- 6

105 + 76

- 22

StemPro 5 x 104 + 23

- 9

105 + 15

- 1

E

F

A

A B

C D

E

C

B

D50

40

30

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10

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o f c o

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i e s

BJ Human Foreskin Fibroblast


ntreated Control Cell Boost reated


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eferences

1. Sommer CA, et al. (2009) iPS cell generation using a singlelentiviral stem cell cassette. Stem Cells 27: 543-549.

2. Somers A, et al. (2010) Generation of transgene-free lungdisease-speci c human iPS cells using a single excisablelentiviral stem cell cassette. Stem Cells 28: 1728-1740.

3. Tchieu J, et al. (2010) Female human iPSCs retain an inactive Xchromosome. Cell Stem Cell 7: 329-342.

4. Buecker C, et al. (2010) A murine ESC-like state facilitatestransgenesis and homologous recombination in humanpluripotent stem cells. Cell Stem Cell 6: 535-546.

5. Huangfu D, et al. (2008) Induction of pluripotent stem cellsfrom primary human broblasts with only Oct4 and Sox2. Nat.Biotechnol. 26: 1269-1275.

6. Ichida JK, et al. (2009) A small-molecule inhibitor of TGF-Betasignaling replaces sox2 in reprogramming by inducing nanog.Cell Stem Cell 5: 491-503.

7. Lin T, et al. (2009) A chemical platform for improved inductionof human iPSCs. Nat. Methods 6: 805-808.

8. Zhu S, et al. (2010) Reprogramming of human primarysomatic cells by Oct4 and chemical compounds. Cell Stem Cell.7:651-655.

9. Li Y, et al. (2010) Generation of iPSCs from mouse broblastswith a single gene, Oct4, and small molecules. Cell Research.1-9.

10. Chan E, et al. (2009) Live cell imaging distinguishes bonade human iPS cells from partially reprogrammed cells. Nat.

Biotechnol. 27:1033-1037.

11. Hockemeyer D, et al. (2008) A drug-inducible system for directreprogramming of human somatic cells to pluripotency. CellStem Cell. 3:346-353.

RELATED PRODUCTS

Description Catalogue No.

Human iPS Cell Boost Supplement SCM088

Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit SCR544

Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit SCR545

FibroGRO Xeno-Free Human Foreskin Fibroblasts SCC058

FibroGRO LS Complete Medium SCMF002

Available from www.millipore.com.

ConclusionIn summary, we have established a robust reprogrammingmethod that is amenable to ef cient reprogramming of different broblasts readily available from human donors.By using small molecules that modulate key signalingpathways and epigenetic modi ers, we could dramaticallyimprove the quality and quantity of human iPS coloniesgenerated.

This novel method increased the number of humaniPS colonies by 2-3 fold when the mouse STEMCCAlentivirus kit was used and up to 15-fold when the humanSTEMCCA lentivirus kit was used. The colonies formedpossessed the distinctive at 2D morphology that aremore reminiscent of human embryonic stem cells andcould be easily passaged in contrast to untreated controlthat exhibited 3D morphology and were more dif cult toisolate. Furthermore, the time to establishment of fully

reprogrammed human iPS colonies that are SSEA4+TRA-1-60+Hoechstdim was signi cantly shortened by 50%.Reprogramming with the small molecule boost supplementwas further validated on multiple human broblast celllines in feeder-free and serum-free culture systems.In summary, the use of the STEMCCA polycistroniclentivirus in combination with the small molecule boostsupplement provides a convenient solution for enhancedreprogramming ef ciency.
http://www.millipore.com/catalogue/item/SCM088&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCC058&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCMF002&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCMF002&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCC058&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR545&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCR544&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SCM088&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RC


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for analyzing invasion at the cell population level, butanalysis of the subcellular events mediating the stages of invasion requires techniques with higher resolution.

The method that has been most informative for pinpointingregions of the cell that initiate invasion involves platingcells on a culture surface coated with a thin layer of

uorescently-labeled matrix, and visualizing regionswhere the cell has degraded the matrix to create an areadevoid of uorescence2. Such assays have revealed thatinvasive cells extend small, localized protrusions thatpreferentially degrade the matrix. These protrusions aretermed invadopodia in cancerous cells, and podosomes innon-malignant cells such as macrophages3. Many moleculesorchestrate the formation and function of invadopodia; afew of the key molecular events include Src phosphorylation

of scaffolding protein Tks54

, N-WASP activation andcortactin regulation of the Arp2/3 complex to induce actinpolymerization5,6, generation of reactive oxygen speciesby NADPH oxidases7, and cortactin-mediated localizationof membrane-type and secreted matrix metalloproteases(MMPs) to the invadopodia8.

EMD Millipores QCM Gelatin Invadopodia Assays providoptimized materials and protocols to enable reproducibleanalysis of invadopodia in invasive tumor cells (Catalog No.ECM670 f or green uorescence, Catalogue No. ECM671forred uorescence). Reagents are provided for coating glassculture surfaces with uorescent matrix and for colocalizingthe actin cytoskeleton and nuclei with invadopodialdegradation sites. This assay may also be used for assessingthe activity of inhibitors and promoters of invadopodiaformation and function. Furthermore, different cell typesand individual cells in heterogeneous populations may beanalyzed for invasive potential. Finally, the assay kits providetroubleshooting suggestions, recommendations for coatingon multiple substrate formats, and example studies in

several assay systems (e.g., various cell types, time-coursestudies, degradation modulation).

Fluorescent Gelatin DegradationAssays for InvestigatingInvadopodia FormationJanet Anderl, Jun Ma and Luke ArmstrongEMD Millipore

AbstractThe invasion of cells through tissue and associatedextracellular matrix is a critical activity in bothphysiological and pathological processes, such asembryological development and cancer metastasis. A

key cellular feature involved in matrix degradation is theformation of protrusions of localized protease activity,termed invadopodia or podosomes. An effective methodfor visualizing subcellular invadopodia formation involvesthe plating of cells onto a thin layer of uorescently-labeled matrix. Areas of degradation are associatedwith a loss of uorescence, and these regions may bemicroscopically imaged and colocalized with molecules of interest in the proteolytic pathway. Here we demonstratethe capabilities of two new invadopodia assay kits, whichprovide the reagents necessary for generating thin coatings

of prelabeled uorescent gelatin on glass substrates.These kits enable simple, rapid, and consistent productionof homogeneous gelatin matrices and visualization of degradation produced by multiple cell types. Furthermore,degradation may be quanti ed by image analysis and usedto characterize proteolytic time-courses and modulatoreffects on invadopodia formation.

IntroductionInvasion of cells through layers of extracellular matrixis a key step in tumor metastasis, in ammation, anddevelopment. Stages of invasion include adhesion to thematrix, degradation of proximal matrix molecules, extensionand traction of the cell on the newly revealed matrix, andmovement of the cell body through the resulting gap inthe matrix1. Each of these stages is executed by a suite of proteins, including proteases, integrins, GTPases, kinases,and cytoskeleton-interacting proteins.

Classical methods for analyzing cellular invasion involveapplication of cells to one side of a layer of gelled matrix

molecules and quantifying the relative number of cells thathave traversed the layer. Such methods are extremely useful
http://www.millipore.com/catalogue/item/ECM670&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM671&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM671&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM670&cid=BIOS-S-EPDF-1189-1108-RC


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poly-L-lysine in deionized water for 20 minutes at roomtemperature (RT). The poly-L-lysine was then removed, andthe slides rinsed three times with 500 L/well of DulbeccosPBS (DPBS). Next, 250 L of dilute glutaraldehyde in DPBSwas added to each well for 15 minutes at RT to activatethe poly-L-lysine surface for further protein attachment.Following removal of the glutaraldehyde, each well wasagain rinsed three times with 500 L of DPBS. Finally, 200

L of gelatin in DPBS, mixed at a 1:5 ratio of uorescently-labeled:unlabeled gelatin, was coated onto each well for 10minutes at RT, followed by three rinses in DPBS. All stepsincluding, and subsequent to, uorescent-gelatin coatingwere performed so as to protect the glass slides fromphotobleaching due to excessive exposure to light.

To prepare for cell plating, the gelatin substrates weredisinfected with 500 L/well of 70% ethanol for 30minutes at RT. After ethanol removal and rinsing in DPBS,free aldehydes were quenched by the addition of 500 L/well of amino-acid-containing growth mediumand incubated at RT for 30 minutes. Cell types of interestwere detached and seeded as described in the previoussection. For some experiments, a modulator of invadopodiaformation, focal adhesion kinase inhibitor II (5 M

nal concentration, or 0.4% DMSO control), was addedsimultaneously with plating.

Sample Fixation and StainingAt the desired time-point after plating, growth medium

was removed from the chamber slides and the sampleswere xed for 30 minutes at RT with 250 L/well of 3.7%formaldehyde in DPBS. Samples were then rinsed twicewith 500 L/well of uorescent staining buffer (DPBSwith 2% blocking serum and 0.25% Triton X-100 for cell

Assay PrincipleThe EMD Millipore QCM Gelatin Invadopodia Assaysprovide the reagents necessary for af xing a thin, uniformlayer of pre-labeled uorescein (green)- or Cy3 (red)-gelatin to a glass culture substrate, allowing for rapiddetection of matrix degradation9,10. A poly-L-lysine coatingis rst adsorbed to the glass substratum. The substrateis then treated with a dilute glutaraldehyde solution tobifunctionally activate the surface for further proteinbinding. Subsequent incubation of the surface with

uorescent gelatin allows covalent coupling between thepoly-L-lysine and gelatin via reactive aldehyde (-CHO)groups. The uorescently-coated glass is now prepared forcell culture by disinfection with 70% ethanol, followed byquenching of free aldehydes with amino acid-containinggrowth medium. Upon completion of uorescent substratepreparation, cell types of interest may be seeded onto thegelatin surface for a desired amount of time. Depending

on cell type, degradation may occur within a few to severalhours, and treatment compounds of interest may also beintroduced within the culture period (Figure 1).

Degraded areas of gelatin, now devoid of uorescence, maybe microscopically visualized and quanti ed using imageanalysis software. The assay also provides uorescently-labeled phalloidin (TRITC- or FITC-conjugated) and DAPI, forvisualization of cytoskeletal F-actin and nuclei, respectively,to allow colocalization of degradation with cellularfeatures. Potential activators or inhibitors of invadopodia

formation may be investigated for their in uence on thedegree and frequency of matrix degradation, and theassay may be further combined with immunocytochemicalstaining for other molecules of interest in mechanisticstudies.

Materials and MethodsCell Lines sedMDA-MB-231 human breast adenocarcinoma, RPMI-7951and SK-MEL-28 human skin melanoma, and IC-21 mouseperitoneal macrophages were obtained from ATCC andcultured to 80-90% con uence in tissue culture asks.For seeding onto gelatin surfaces, cells were detachedusing 0.25% trypsin-EDTA (or DPBS without calcium andmagnesium for IC-21 cells), pelleted, then resuspendedin growth medium to a concentration of 28,000 cells/mL(20,000 cells/cm2). Cells were seeded in a volume of 500 L/well and cultured for the desired duration of degradation, generally between 8-48 hours.

Substrate Preparation and Cell Seeding

To facilitate attachment of uorescent gelatin, 8-well glasschamber slides were rst coated with 250 L/well of dilute

Figure 1. Gelatin invadopodia assay setup.

3. Fluorescent Gelatin

(10 min RT)

4. 70% Ethanol (30 min RT)

5. Growth Medium (30 min RT)

6. Cell ype/ reatmentof Interest in GrowthMedium (e.g 24 hr, 37 C)

2. Glutaraldehyde(15 min RT)

1. Poly-L-Lysine(20 min RT)

Glass Substrate

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O

C H O


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Imaging and AnalysisMounted cover glasses were allowed to hard-set before

uorescent imaging with illumination and ltersappropriate for uorescein/FITC, Cy3/TRITC and DAPIexcitation and emission wavelengths. Samples were imagedon an inverted wide- eld uorescent microscope at 20Xobjective magni cation for quanti cation studies (5 eldsof view per well) or at 63X objective magni cation (oil

immersion) for colocalization experiments.

Image analysis was performed using free, downloadableImageJ software distributed by the National Institutesof Health (NIH)10. A high intensity threshold was set forpositive DAPI signal, then analyzed as particles fordetermination of a nuclear (cell) count. Similarly, settinga high intensity threshold for phalloidin signal enabledmeasurement of total cell area per eld of view. Conversely,a low intensity threshold was set for diminished

uorescent gelatin signal to enable quanti cation of totaldegradation area per eld of view (Figure 2).

esults and DiscussionWe demonstrated the ability to visualize and quantify thedegradation of uorescent gelatin matrices by a varietyof cell types. For the cell lines MDA-MB-231, RPMI-7951,and IC-21, gelatin proteolysis demonstrated a range of degradation patterns that may be attributed to invadopodiaor podosome formation, including punctate, linear, orblotchy areas devoid of uorescein-gelatin uorescence

(Figure 3). Often, not all cells in a population will exhibitproteolytic behavior, and cellular movement between sitesof degradation may frequently be observed. SK-MEL-28cells, a noninvasive melanoma type, did not display gelatindegradation (Figure 3).

permeabilization). For immunocolocalization studies, 200 Lof primary antibody in uorescent staining buffer was addedto each well for 1 hour incubation at RT. Samples were thenrinsed three times with 500 L/well of uorescent stainingbuffer before proceeding on to 1 hour RT incubation with

uorescent secondary antibody, uorescently-conjugatedphalloidin (2 g/mL) and DAPI (1 g/mL) in staining buffer.Primary and secondary antibodies were omitted for stainsincorporating phalloidin and DAPI only. Finally, samples wererinsed twice each with uorescent staining buffer and DPBSbefore removal of culture chambers and cover-slipping.Slide-mounting medium contained anti-fade reagent and

appropriately thick cover glasses were selected for imagingmagni cation of choice.

Figure 3. Fluorescent gelatin degradation and phalloidin/DAPI staining of multiple cell types. Fluorescein-gelatin matrices (toppanel, green) were coated onto 8-well glass chamber slides. Multiple human cancer cell lines (MDA-MB-231, RPMI-7951, and SK-MEL-28) and a mouse macrophage cell line (IC-21) were plated onto the gelatin substrates at 20,000 cells/cm2 for a culture durationof 24 hours. F-actin and nuclei were stained, respectively, with TRITC-phalloidin (bottom panel, red) and DAPI (bottom panel, blue).Cells were imaged at 63X objective magni cation (bar = 25 m).

DAPI Nuclear Count

hreshold

Count

Phalloidin otal Cell AreaFluorescent GelatinDegradation Area

hreshold andArea Measurement

hreshold andArea Measurement

Figure 2. Example imageanalysis with NIH ImageJsoftware.

MDA-MB-231(Human Breast

Adenocarcinoma)PMI-7951

(Human Skin Melanoma)SK-MEL-28

(Human Skin Melanoma)

IC-21(Mouse Peritoneal

Macrophage)

Fluorescein-Gelatin

I C-Phalloidin/DAPI


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Colocalization StudiesIn Figure 4, RPMI-7951 cells seeded onto Cy3-gelatinmatrices demonstrated the ability to co-localize sites of gelatin degradation with phalloidin (F-actin) puncta andcortactin foci. Cortactin protein is strongly associated withactin assembly, and colocalization of this molecule withareas of proteolysis was indicative of dynamically activeinvadopodia formation (see white arrows in gure 4).

ime-dependent DegradationOver 100 cells per condition were analyzed to obtain thepercent degradation area of total cell area data depictedin Figure 5. For MDA-MB-231 and IC-21 cells, degradationpercentage increased over time (particularly between 8and 24 hour time points), whereas no degradation bynoninvasive SK-MEL-28 cells was observed. Of note is thatalthough the theoretical maximum of percent degradationarea of total cell area is 100%, higher amounts of degradation were observed here, likely due to cellularmovement during proteolysis. Such historical degradationis recorded using this assay, resulting in degradation areaslarger than the area of a cell itself (particularly for longertime points or highly motile cell types).

Modulation of Matrix DegradationCells were seeded onto uorescein-gelatin matrices andsimultaneously treated with focal adhesion kinase (FAK)inhibitor II (PF-573,228) or a DMSO control (Figure 6). FAKinhibition, which has previously been shown to enhance

invadopodia formation in certain cell types12, was indeedobserved to increase MDA-MB-231-associated degradationover the course of 24-hour treatment. The non-invasivephenotype of SK-MEL-28 cells was not altered by additionof FAK inhibitor II, but surprisingly, degradation by IC-21cells was decreased by treatment with the compound. Suchopposite effects as those seen between the MDA-MB-231and IC-21 cells emphasize variations in proteolytic behaviorbetween cell types, and may be indicative of signi cantdifferences in degradation signaling mechanisms betweencancerous and normal cell phenotypes.

Figure 4. Colocalization of degradation with invadopodia-related puncta.Cy3-gelatin matrices were coated onto glass chamber slides, and RPMI-7951 human skinmelanoma cells were seeded onto the gelatin substrates for 24 hours. For uorescentimmunocytochemistry, cells were incubated with anti-cortactin, followed by detection with a Cy5-conjugated secondary antibody. Secondary antibody incubation was performed concurrently withFITC-phalloidin and DAPI staining. Cells were imaged at 63X objective magni cation.

Figure 5. ime-dependent gelatin degradation. Multiple cell types (images are of MDA-MB-231)were plated onto Cy3-gelatin substrates (top image panel, red) and cultured for 8, 24, or 48hours. Following staining with FITC-phalloidin (bottom image panel, green) and DAPI (bottomimage panel, blue) cells were imaged at 20X objective magni cation at 5 elds of view per well.Bar = 100 m. Percent degradation area of total cell area was quanti ed using ImageJ analysissoftware, as depicted in Figure 2.

DAPI Cy3-Gelatin

FI C-Phalloidin Cy5-Cortactin Antibody Gelatin/ Phalloidin Overlay

FI C-Phalloidin/DAPI

125

100

75

50

25 % D

e g r a

d e d A r e a

Cy3-Gelatin: % Degradation Area of Total Cell Area

0MDA-MB-231 SK-MEL-28

8 hr

24 hr

48 hr

Cell Type

IC-21

8 hr 24 hr 48 hr

Cy3-Gelatin


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ConclusionOur results demonstrate the utility of EMD MilliporesQCM Gelatin Invadopodia Assays in the visualization andquanti cation of gelatin degradation by a variety of celltypes at multiple time points and following treatmentwith modulators of invadopodia formation. Speci cally, weshow that both time-dependence of matrix degradationand ef cacy of invadopodia modulators are heavilyin uenced by cell type, inviting further, detailed studiesof the differential signaling controlling these processes.To this end, QCM Gelatin Invadopodia Assays can providea convenient, exible system for monitoring matrixdegradation and investigating key players in the proteolyticprocess, at the single cell and subcellular levels.

Figure 6. Modulation of gelatin degradation by a FAK inhibitor. Fluorescein-gelatin matrices (top image panel, green) were seededwith MDA-MB-231, SK-MEL-28, or IC-21 cells and simultaneously treated with 5 M FAK inhibitor II or a 0.4% DMSO control.Following 24 hour treatment, cells were xed and stained for F-actin and nuclei with TRITC-phalloidin (bottom image panel, red) andDAPI (bottom image panel, blue). Samples were imaged at 20X objective magni cation at 5 elds of view per well. Bar = 100 m.

ORDER INFO & RELATED PRODUCTS


QCM Gelatin Invadopodia Assay (Green) ECM670

QCM Gelatin Invadopodia Assay (Red) ECM671

Mouse Anti-Cortactin (p80/85), Clone 4F11 05-180

Mouse Anti-MMP-14 [MT1-MMP],Clone LEM-2/15.8

MAB3328

Mouse Anti-Src, Clone GD11 05-184

Rabbit Anti-Tks5 (SH3 #1) 09-403

Rabbit Anti-N-WASP AB2962

Rabbit Anti-Arp2 AB3886


Rabbit Anti-Arp3 07-272

GM6001 [Ilomastat] MMP Inhibitor CC1100

0.25% Trypsin-EDTA in Hanks BalancedSalt Solution

SM-2003-C

EmbryoMax 1X Dulbeccos PhosphateBuffered Saline

BSS-1006-B

Available from www.emdbiosciences.com.

Focal Adhesion Kinase Inhibitor II 324878

PP2 (Src Inhibitor) 529573


eferences1. Friedl P and Wolf K. Plasticity of cell migration: a multiscale

tuning model. J Cell Biol 2010; 188:11-19.2. Chen WT et al. Expression of transformation-associated

protease(s) that degrade bronectin at cell contact sites. J CellBiol 1984; 98:1546-1555.

3. Ayala I et al. Invadopdia: a guided tour. Eur J Cell Biol 2006;85:159-164.

4. Seals DF et al. The adaptor protein Tks5/Fish is required forpodosome formation and function, and for the protease-driven invasion of cancer cells. Cancer Cell. 2005; 7:155-165.

5. Yamaguchi H et al . Molecular mechanisms of invadopodiumformation: the role of the N-WASP-Arp2/3 complex pathwayand co lin. J Cell Biol 2005; 168: 441452.

6. Weaver AM. Invadopodia: specialized cell structures for cancerinvasion. Clin Exp Metastasis. 2006; 23:97-105.

7. Diaz B et al. Tks5-dependent, Nox-mediated generationof reactive oxygen species is necessary for invadopodiaformation. Sci Signal 2009; 2:ra53.

8. Clark ES and Weaver AM. A new role for cortactin ininvadopodia: regulation of protease secretion. Eur. J. Cell Biol2008; 87: 581-590.

9. Artym VV, Yamada KM and Mueller SC. ECM degradationassays for analyzing local cell invasion. Methods Mol Biol2009; 522:211-219.

10. Xu X, Johnson P and Mueller SC. Breast cancer cell

movement: imaging invadopodia by TIRF and IRM microscopy.Methods Mol Biol. 2009; 571: 209-225.

11. Rasband WS (1997-2011), ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/.

12. Liu S. et al. Laminin-332-b1 integrin interactions negativelyregulate invadopodia. J Cell Physiol. 2010; 223:134-142.

MDA-MB-231(DMSO Control)

MDA-MB-231(5 M FAK Inhibitor II)

IC-21(DMSO Control)

IC-21(5 M FAK Inhibitor II)

Fluorescein-Gelatin

I C-Phalloidin/DAPI

40

30

20

10 %

D e g r a

d e d A r e a

Fluorescein-Gelatin:% Degradation Area of Total Cell Area

0MDA-MB-231 SK-MEL-28

DMSO Control

5 M FAK Inhibitor II

Cell TypeIC-21
http://www.millipore.com/catalogue/item/ECM670&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM671&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/05-180&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/MAB3328&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/05-184&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/09-403&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/AB2962&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/AB3886&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/07-272&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/CC1100&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SM-2003-C&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/BSS-1006-B&cid=BIOS-S-EPDF-1189-1108-RChttp://www.emdchemicals.com/life-science-research?WT.mc_id=5USEN1059http://www.emdchemicals.com/life-science-research?WT.mc_id=5USEN1059http://www.emdchemicals.com/life-science-research?WT.mc_id=5USEN1059http://www.emdchemicals.com/products/EMD_324878?WT.mc_id=5USEN1059http://www.emdchemicals.com/products/EMD_529573?WT.mc_id=5USEN1059http://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RChttp://www.emdchemicals.com/products/EMD_529573?WT.mc_id=5USEN1059http://www.emdchemicals.com/products/EMD_324878?WT.mc_id=5USEN1059http://www.emdchemicals.com/life-science-research?WT.mc_id=5USEN1059http://www.millipore.com/catalogue/item/BSS-1006-B&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/SM-2003-C&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/CC1100&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/07-272&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/AB3886&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/AB2962&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/09-403&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/05-184&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/MAB3328&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/05-180&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM671&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/ECM670&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/?cid=BIOS-S-EPDF-1189-1108-RC


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Bead-based assays, such as those using LuminexxMAP technology, have enabled the measurement of phosphorylation levels of up to 12 proteins simultaneously.EMD Millipores MILLIPLEXmap Akt/mTOR Panel is abead-based immunoassay that simultaneously detects 11phosphoproteins (Figure 1) in the Akt/mTOR pathway ina single cell lysate sample, enabling the measurement of phosphorylation changes in this important pathway.

Here, we demonstrate the utility of the MILLIPLEXmap Akt/mTOR 11-plex Panel in the analysis of Akt/mTORsignaling in cancer cell lines (HepG2, HEK293, and MCF7).All analytes were detected with good speci city, sensitivityand precision. In addition, we demonstrate successfuldetection of these phosphoproteins in human as well as

mouse tissue samples (with the exception of phospho-IGF1R in mouse samples). Finally, our inhibitor studiesshow the utility of this panel in drug discovery research.

Exploring Akt/mTOR SignalingUsing the MILLIPLEXmap Akt/mTOR 11-plex PanelJoseph Hwang, Ph.D.EMD Millipore

IntroductionThe Akt signaling pathway, one of the most often-dysregulated signaling pathways in cancer, plays animportant role in mediating a very broad range of cellularprocesses such as growth and development, cell cycle,

energy homeostasis and survival. Akt, a serine/threoninekinase that phosphorylates over 100 protein substrates,mediating cell survival and proliferation signals, is oftenitself hyperphosphorylated in various tumor types.Although the Akt gene itself is not known to be mutatedin cancers, many of the upstream regulators of Akt,including IR, IRS, PI3K and PTEN, are oncogenes and tumorsuppressors. Downstream of Akt, the mammalian Target Of Rapamycin (mTOR) complex is a key regulator of growthand metabolism.

As nearly all of the players in the Akt/mTOR signalingpathway are coordinately regulated by phosphorylation,understanding the role of this pathway in normalphysiological processes and in diseases such as cancer anddiabetes requires the ability to simultaneously measurephosphorylation status of multiple protein targets.Several assays to examine phosphorylation status arecurrently available, including Western blotting, ELISA,reverse phase arrays, quantitative cell imaging, and massspectroscopy. Although some of these platforms yieldabsolute, quantitative data, the assays are either limitedto measuring only one analyte at a time, or are excessivelydif cult or expensive.

On the level of multiparametric single-cell analysis, owcytometry has enabled the study of multiple pathwayactivation and cross-talk in a time-dependent manner.Directly applicable to the Akt/mTOR pathway, EMDMillipores FlowCellect PI3K-mTOR Assay Kit uses directlyconjugated antibodies against phospho-Akt1/PKB (Ser473)and phospho-ribosomal protein S6 (Ser235) to analyze

both upstream and downstream portions of this signalingcascade.

Figure 1. he Akt/m O signaling pathway. Analytes included inthe MILLIPLEXmap Akt/mTOR 11-plex Panel are highlighted in red.

I R

I R

GSK3

Akt

TSC2

TSC1

PIP3

PIP2

Translation Initiation

Rheb

PRAS40

mTOR

p70S6K

RPS6

I R S P 1

3 K

PDK1

4 E-BPI

PDCD4

P T E N

eIF4B

eIF2B

e IF4E

eIF 4A


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by bicinchoninic acid (BCA) assay of sample aliquotswe observed typical protein recoveries of 5-10% of initial tissue mass. After diluting samples to 2 mg/mL inlysis buffer, they were transferred to 96-well plates inpreparation for assay with the MILLIPLEXmap kit.

Immunoassay ProtocolThe multiplex assay was performed in a 96-well plate

according to product instructions supplied for theMILLIPLEXmap Akt/mTOR 11-plex Panel (EMD MilliporeCat. No. 48-611).The plate was rst rinsed with 100 Lassay buffer. 25 L of controls and samples and 25 Lbeads were added to each well. Plates were incubatedovernight at 4 C (alternatively can be incubated 2 hoursat room temperature (RT)). Beads were washed twice withassay buffer, then incubated 1 hour at RT with biotinylateddetection antibody cocktail. The detection antibody cocktailwas replaced with 25 L streptavidin-phycoerythrin (SAPE)and incubated for 15 minutes at RT. 25 L of ampli cationbuffer was added and incubated another 15 minutes atRT. Finally, the SAPE/ampli cation buffer was removedand beads were resuspended in 150 L assay buffer. Theassay plate was read and analyzed in a Luminex 200system. This is a compact unit consisting of an analyzer, acomputer, and software (Luminex Corporation, Austin, TX).

esults and DiscussionThe MILLIPLEXmap Akt/mTOR 11-plex Assay providedhigh speci city, indicated by the detection of proteins

at the expected molecular weights as shown byimmunoprecipitation/Western blot (Figure 2A). Speci citywas also demonstrated by detection of the correctisoforms of GSK3a/b and IR/IGF1R (Figure 2B). In addition,analytical validation experiments (data not shown; detailsavailable on www.millipore.com) demonstrated high signal-to-noise ratios, sample linearity, and precision, lendingsupport to the robustness of this kit.

All analytes in the MILLIPLEXmap Akt/mTOR 11-plexPanel were detected in human and mouse tissues usingthe kit, with the exception of phospho-IGF1R, which ishuman-speci c. Of interest is the observation that bothbreast cancer patients exhibited greater than a 2-foldincrease in phosphorylation of mTOR compared to breasttissue from healthy subjects (Figure 3). This observationis consistent with cancer cells exhibiting a higher levelof protein synthesis than normal cells. However, Akt,which is upstream of mTOR, and p70S6K and RPS6, bothdownstream of mTOR, did not exhibit a signi cant changein phosphorylation levels.

The addition of wortmannin, an inhibitor of PI3K,resulted in decreased levels of phosphorylation of several

Methodsissue Culture

HepG2, HEK293, and MCF7 cells were cultured according toATCC guidelines in recommended media. Cells were grownto approximately 85% con uence, then serum starved for 4hours prior to treatment with stimuli or compounds.

Sample PreparationCells were lysed and samples collected according to theMILLIPLEXmap Cell Signaling Buffer and Detection Kit (EMDMillipore Cat. No. 48-602) instructions.

MicrospheresWe developed the MILLIPLEXmap Akt/mTOR 11-plex Panel byconjugating speci c capture antibodies to microsphere beadspurchased from Luminex Corporation. Each set of beadsis distinguished by different ratios of two internal dyes,yielding a unique uorescent signature to each bead set.

Human and Mouse issue HomogenizationFrozen tissue samples were weighed and placed on ice.Samples were homogenized with 1 mL lysis buffer per50-100 mg mouse tissue or 1 mL lysis buffer per 30-50mg of human tissue. Tissues were homogenized using theOmni International General Laboratory Homogenizer withOmni Tip- Plastic Generator Probes at setting level 2for 30 seconds. Samples were then incubated with gentlerocking at 4 C for 15 minutes and centrifuged (10,000g for 10 minutes at 4 C) to separate connective tissue,

fat, ECM, etc. Supernatants were placed in new tubes.Protein concentration in each sample was determined

Figure 1. Speci city of the MILLIPLEXmap Akt/m O 11-plex Panel.Phosphorylated proteins weresimultaneously detected indifferent cell lines treatedwith either insulin or IGF1.(A) Immunoprecipitation (IP)of phosphoproteins wereperformed with capture beadsand detected by Westernblotting with the biotinylateddetection antibodies. (B)Isoform cross-reactivity testswere performed using humanrecombinant GSK3a/b byWestern blotting or IR/IGF1Rby IP/Western blotting.

B. Cross-reactivity

GSK3a/b Cross-reactivityGSK3

IR/IGF1R Cross-reactivity

(IP/Western)

a b a b

HEK293 NIH3T3/IGF1R NIH3T3/IR

HEK293 NIH3T3/IGF1R NIH3T3/IR

IGF1

IGF1

PIR

PIGF1R

A. IP/Westerns

pp70S6K

pAkt

NT Ins NT IGF NT IGFNT IGF NT SerumNT Ins

pIRS1

pPTEN

pGSK3a

pIR

pIGF1R

pRPS6

pGSK3b

pTSC2 pmTOR

(HEPG2) (HEK293) (HEK293) (HepG2) (HEK293) (HEK293)

NT EGF NT IGFNT IGF NT IGFNT IGF(A431) (HEK293) (MCF-7) (HEK293) (MCF-7)
http://www.millipore.com/catalogue/item/48-611&cid=BIOS-S-EPDF-1189-1108-RChttp://www.millipore.com/catalogue/item/48-611&cid=BIOS-S-EPDF-1189-1108-RC


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downstream targets such as Akt, mTOR, p70S6K and RPS6(Figure 4). Rapamycin, an mTOR inhibitor, also inhibitedits downstream targets p70S6K and RPS6. Consequently,these studies demonstrate that tumor development iscomplex and involves more than the dysregulation of a

single pathway. This conclusion further underscores theimportance of simultaneous measurement of multiplephosphoprotein targets and demonstrates the utility of theMILLIPLEXmap Akt/mTOR 11-plex Panel.

Figure 3. Phosphorylated Akt/mTOR pathway proteins were simultaneously detected in human (A) and mouse (B) tissue samples. (20 g/mL). Total Akt/mTOR proteinswere detected simultaneously in mouse (C) tissue samples using the MILLIPLEXmap Total Akt/mTOR 11-plex Panel (available late 2011). Human matched breast normaland cancer tissue samples were purchased from Asterand. Mouse tissues from C57BL/6J males were purchased from Jackson Laboratory. Values were background-subtracted and reported as mean uorescence intensity (MFI). ND represents not detectable.

Figure 4. PhosphorylatedAkt/m O pathway proteinswere detected simultaneouslyin HepG2 cells treated withvarious inhibitors . Cellswere pretreated with 0.1M wortmannin, 0.1 Mrapamycin, 10 M U0126(MEK1/2 inhibitor), 50 MLY-294002 (PI3K inhibitor), orRo-31-8220 (PKC and GSK3b inhibitor) for 30 minutes priorto the addition of 10 g/mLinsulin for 15 minutes. Valueswere background-subtractedand reported as percent of vehicle and insulin-treated

values, respectively.eferences

1. Khan, I et al. Assay and DrugDev Tech 2010, 8(1), 27-36.

2. Alvarex, R et al. G. J ClinOncology 2010, 28(20),3366-79.

3. Crowell, J et al. Mol CancerTher. 2007, 6(8), 2139-48.

4. Wang, X and Proud, C.Physiology 2006, 21, 362-9.

5. Vignot, S et al. Annals of Oncology 2005, 16, 525-37.

RELATED PRODUCTSDescription Catalogue No.

MILLIPLEXmap 11-plex Akt/mTOR Panel 48-611

MILLIPLEXmap Cell Signaling Bufferand Detection Kit

48-602

IR (Tyr1162/Tyr1163) MAPmate 46-688

IRS1 (pan Tyr) MAPmate 46-627

Akt (Ser473) MAPmate 46-677

PTEN (Ser380) MAPmate 46-679

TSC2 (Ser939) MAPmate 46-691

mTOR (Ser2448) MAPmate 46-686GSK3b (Ser9) MAPmate 46-690


p70S6K (Thr424) MAPmate 46-629

Total IR MAPmate 46-687

Total Akt MAPmate 46-675

Total PTEN MAPmate 46-678

Total mTOR MAPmate 46-685

Total GSK3b MAPmate 46-689

Total p70S6K MAPmate 46-630

FlowCellect PI3K-mTOR Signaling Cascade Kit FCCS05210

FlowCellect PI3K Activation Dual Detection Kit FCCS025105


M F I

M F I

Phosphorylated Protein p 7 0 S 6 K

I R S 1

G S K 3

I G F 1 R

G S K 3

A k t I R

T S C 2

m T O R

Breast normal 1

Breast cancer 1Breast normal 2

Breast cancer 2

600

500

400

300

200

100

0

PTEN RPS6

15000

10000

5000

0

Mouse Tissues p70S6K IRS1 GSK3a IGF1R GSK3b Akt PTEN IR RPS6 TSC2 mTORColon ND 27 23 ND 42 ND 11094 53 7 4 NDPancreas 305 116 198 ND 62 215 10050 136 676 212 81Pituitary 28 171 112 ND 176 198 10956 60 967 540 1183Prostate 143 326 35 ND 176 163 10906 82 345 454 1102SkeletalMuscle 186 665 38 ND 552 15 6643 12 265 374 636

Testicles 250 108 101 ND 57 52 13303 122 133 1346 1513Liver 80 130 565 ND 51 226 13630 101 5696 562 458

MouseTissues p70S6K IRS1 GSK3a IGF1R GSK3b Akt PTEN IR RPS6 TSC2 mTORColon 157 61 25 ND 59 84 51 163 39 86 50Pancreas 188 87 35 ND 37 140 51 220 50 82 61

Pituitary 2173 5434 2722 ND 269 2212 1752 509 241 370 305Prostate 1556 5382 2673 ND 233 2309 1771 475 214 367 317SkeletalMuscle 1324 4181 2704 ND 749 3475 2035 528 92 789 228

Testicles 1222 4241 2232 ND 619 3091 1821 426 85 608 171Liver 1146 5387 2421 ND 160 2145 794 525 54 240 246

C. Total Akt/mTOR proteins in mouse tissue samples

B. Phosphorylated Akt/mTOR proteins in mouse tissue samplesA. Phosphorylated Akt/mTOR proteins in human breasttissue samples

% C

o n t r o l

S i g n a l

Phosphorylated Protein

p 7 0 S 6 K

( T h r 4

2 4 )

I R S 1

( S e r

3 1 2 )

G S K 3

( S e r

2 1 )

I G F 1 R ( T y r

1 1 3 5 /

T y r 1

1 3 6 )

G S K 3

( S e r

9 )

A k t

( S e r

4 7 3 )

P T E N

( S E R 3 8 0 )

I R ( T y r

1 1 6 2 /

T y r 1

1 6 3 )

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Immuno-monitoring Using theScepter 2.0 Cell Counter andSoftware ModuleAmedeo Cappione, Ph.D.1; Emily Crossley2; Nagaraja hirumalapura, DVM, Ph.D.3; and Debra Hoover, Ph.D.11EMD Millipore;2Dept. of Pathology, Dept. Microbiology & Immunology, niversity of exas Medical Branch;3Dept. of Pathology, niversity of exas Medical Branch

AbstractBiological samples such as primary isolates or culturedcells are often heterogeneous mixtures of cells thatdiffer by type and/or function. Such differences are mostcommonly determined by multicolor uorescent antibody

detection of speci c cell markers using ow cytometry. Inaddition to variations in protein expression, many cell typesand physiological states are also distinguishable by size.The ability to identify subsets on the basis of phenotypicdifferences and determine their relative frequencies (andconcentrations) is critical to many aspects of research.

The Scepter cell counter combines the ease of automatedinstrumentation and the accuracy of impedance-basedparticle detection in an affordable, handheld format. Usinga sensor with a 40 m aperture, the Scepter cell counter

can accurately and precisely count a broad range of celltypes, including small cells (> 4 m in diameter) andperipheral blood mononuclear cells (PBMC)1. This articleoutlines three examples of experiments using the Sceptercell counters sensitive size-discriminating capability todemonstrate rapid, qualitative assessment of individual cellpopulation frequencies in complex cell mixtures.

Exa E 1:Lymphocyte vs. monocyte subset

01discrimination in human PBMCs

IntroductionThe human immune system is comprised of cell subsetswith distinct functional pro les that ght pathogens.Assessing pro les of the various subsets, such aslymphocytes and monocytes, can help identify molecularsignatures that may facilitate research, ranging fromvaccine development to prognostic advancements.TheScepter cell counter, in combination with Scepter SoftwarePro, provides a tool for rapid determination of lymphocyteand monocyte concentrations in PBMC isolates.

Materials and MethodsHuman blood sample prepHuman PBMCs were isolated from whole blood of healthy donors by Ficoll-Paque density centrifugation(GE Healthcare). Brie y, 9 mL of blood was diluted to 25

mL with phosphate-buffered saline (1X EmbryoMax PBS,EMD Millipore) and layered over 15 mL of Ficoll. Sampleswere centrifuged at 400 x g for 30 minutes with no brake,and the resulting PBMC layer was recovered. The PBMCfractions were washed twice with PBS prior to analysis.

Scepter cell countingThe Scepter cell counter was used to count samplesfollowing the simple on-screen instructions. Brie y, theuser attaches a 40 m sensor, depresses the plunger,submerges the sensor into the sample, then releases the

plunger drawing up 50 L of cell suspension. The Sceptercell counter detects each cell passing through the sensorsaperture, calculates cell concentration, and displays ahistogram of cell diameter or volume. Test les wereuploaded and analyzed using Scepter Software Pro todetermine the concentrations and relative cell frequenciesfor the lymphocyte and monocyte fractions.

Cell counting and viability determination using Guava ViaCount assay10 L samples were mixed with 190 L ViaCount reagent,incubated for 5 minutes at room temperature (RT). Sampledata was acquired on a guava easyCyte instrument andanalyzed using guava ExpressPro software.

Cell surface staining and subset determinationFor each sample,100,000 PBMCs were resuspended in 100L PBS+0.1% bovine serum albumin (BSA). Samples werestained with the following combination of uorescentlylabeled antibodies: CD3-PE (T cells), CD19-Alexa Fluor488 (B cells), CD16/CD56-APC (NK cells), and CD14- PEC


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(Monocytes) (antibodies all from eBioscience). Singlestain and isotype controls were included to ensure properinstrument setup. Samples were incubated at RT for 20minutes, washed twice with PBS, then resuspended in200 L PBS prior to acquisition. Samples were analyzed(3000 cells/sample well) on a guava easyCyte system usingguava ExpressPro software.

esults Viability assessment of PBMCFreshly prepared or frozen PBMC samples consist of livecells, dead cells, and a considerable amount of cellulardebris. To understand the relative proportions, freshlyisolated PBMC were stained with ViaCount reagent. Thereagent consists of a cell-permeant nuclear dye that stainsall nucleated cells, and a cell-impermeant dye thatbrightly labels dying cells. Cellular debris is not stained byeither dye.

In the example shown, more than 95% of the cells wereviable. The three components were also distinguishableby particle size. The histograms in Figure 1B show thedistribution of particles in the different fractions asfunction of forward scatter, a ow-based correlate of

particle size. While overlap exists between dead cells anddebris, live cells constituted a fraction made up of twodifferently-sized cell types.

PBMC subset enumeration( cells, B cells, NK cells, monocytes)PBMC can be subdivided into many distinct cell typesbased on expression of speci c surface markers. The

abundance and relative distribution of these subsets arefunctions of developmental state as well as overall health.Two main PBMC populations are lymphocytes (CD14-) andmonocytes (CD14+). Lymphocytes can be subdivided into Tcells (CD3+), B cells (CD19+), and NK cells (CD16/56+).

To examine the ability of 40 m sensors to discriminatelymphocytes from monocytes, PBMC fractions wereanalyzed by both a guava easyCyte and Scepter cellcounter. Representative plots are presented in Figure3. In each case, three main peaks were identi ed byeach analysis method, with greater peak resolution inthe ow cytometry data, particularly in the separationof lymphocytes from debris. The peaks correspond tolymphocytes (small cells), monocytes (large cells), and adebris/dead cell fraction.

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Figure 1. Size-baseddiscrimination of live vs deadcells. (A) live cell (blue), dead

cell (red), and debris (green)fractions were de ned using ViaCount reagent. (B) The fourhistograms show the relativedistribution of total eventsand each gated fraction.

Figure 2. Multicolor owcytometric analysis of PBMCfractions. (A) Live cells (red)are distingui shed from debris/dead cells (green). (B) Livecells are fractionated basedon expression of CD14, CD3,CD16/56, and CD19. (C) Thefour histograms show thelocalization of each subsetto either of the two peaksde ned by forward scatter.


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Across the nine PBMC samples analyzed (Table 1),the average mean cell diameters were 7.230.30 mand 10.020.20 m for lymphocytes and monocytes,respectively. Values were consistent with previouslyreported size ranges2. Cell frequencies were alsodetermined by three methods: Scepter diameter plot,guava easyCyte forward scatter, and antibody staining.The Scepter values slightly underestimated the lymphocytefraction while overestimating the monocyte subset. This islikely the result of the Scepter cell counters comparativelylower resolution as well as subjectivity and user bias ingating. Overall, there was good agreement between the

different analytical techniques with values varying by


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with anti-CD3 mAb (clone HIT3a; BD Pharmingen, 10 g/mL). Mitogenic stimulation was carried out in the presenceof 2 g/mL PHA (Sigma). Unstimulated control cultureswere also analyzed.

Scepter cell countingSample acquisition and data analysis was performed aspreviously described. Test les were analyzed using Scepter

Software Pro to determine the degree of cell activation aswell as concentrations for both unstimulated and activatedfractions.

CD25 staining for activation Following two-day stimulation, cultures were harvested,washed twice with PBS, counted, and stained as previouslydescribed. For each culture, 100,000 cells were resuspendedin 100 L PBS+0.1% BSA. To distinguish the activated T cellfraction, samples were stained with anti-CD3-PE ( T cells)and anti-CD25-APCeFluor780 (eBioscience). Samples wereanalyzed (3000 cells/sample well) on a guava easyCyte HTsystem using guava ExpressPro software.

esultsTo evaluate utility of the Scepter cell counter for rapidqualitative monitoring of immune cell activation,PBMCs were stimulated in culture using two differentmechanisms: (1) CD3/CD28 Ab-mediated co-stimulationof the TCR and (2) stimulation by PHA. CD25 expressioncorrelated with an increase in cell size (Figure 4A). Figure4B shows three main populations of cells: resting T cell(CD3+/CD25-), activated T cells (CD3+/CD25+), and nonT cells (CD3-/CD25-). By comparison, cultures stimulatedwith CD3/CD28 Ab showed greater levels of T cell activationthan those exposed to PHA. Control cultures displayed verylow frequencies of activated cells..

Figure 4C-D compares the Scepter cell counters abilityto detect activation to that of the guava easyCyteplatform. While the Scepter cell counter clearly detectedthe larger, activated cell fraction in stimulated cultures,

the device was unable to simultaneously discriminatethe unstimulated population. This was most likely due tothe presence of a large number of overlapping dead cells

Figure 4. CD25 expression correlates with size increase. (A) An elliptical gate was used to identify live cells. CD25+ cells are red. Live cells arefractionated using CD3 and CD25 Ab. (B) The two histograms in each row show the relative distribution of each fraction as a function of particle size(forward scatter). Plots are based on total events and live cells, respectively. (C) Scepter histogram data for each sample.

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(green cells, Fig. 4A). This was clearly demonstrated in theuntreated sample--two peaks were seen in the histogramplot of total cells (Marker R5). By comparison, the live cell-only histogram showed a single peak within R5.

Exa E 3: 03Splenic cell shift in murine model of Ehrlichiosis

IntroductionHuman monocytotropic ehrlichiosis (HME) is a tick-bornedisease caused by the obligately intracellular pathogenEhrlichia chaffeensi. HME manifests as nonspeci c u-like symptoms but can progress to life-threatening toxicshock-like syndrome with anemia, thrombocytopenia,and multi-organ failure. Extensive in ammation in theabsence of bacterial burden suggests that mortality is dueto unregulated Immunopathology5. In this study, a murineHME model based onEhrlichia muris infection of C57BL/6mice was used to investigate the immune response, inparticular, changes to splenocyte population dynamics.

Materials and methodsInfection of mice with Ehrlichia muris

Six- to eight-week-old C57BL/6 mice were infectedintraperitoneally withEhrlichia muris (~1 x 104 genomes).

Mice were sacri ced on day 30 post-infection andspleens harvested. Single-cell suspensions were preparedusing the GentleMACS Tissue Dissociator following themanufacturers instructions (Miltenyi Biotec Inc., CA).

Determination of splenocyte cell distribution using theScepter cell counterCells were diluted (~1 2 x 105 cells/mL) and acquired

using the Scepter cell counter and 40 M sensor. Gateswere set to exclude red blood cells using an uninfected,nave mouse sample. Splenocytes from three uninfectedmice and three mice infected withEhrlichia muris wereused. Data were analyzed using Scepter Software Pro.

Determination of splenocyte cell distributionby ow cytometrySplenocytes were incubated with Near IR LIVE/DEADFixable Dead Cell Stain Kit (Invitrogen, CA) for 10 minutesin PBS at RT. Cells were washed, blocked with Fc Block(BD Biosciences, CA), puri ed rabbit, rat, and mouse IgGs(Jackson ImmunoResearch, PA) then stained with anti-B220-PerCPCy5.5 (BD Biosciences, CA) and anti-Ter119-APC (eBioscience, CA) and analyzed by ow cytometry.

Figure 5. Forward vs. side scatter plots reveal the presence of two main splenocyte populations. (A,B) The fraction containing larger cells is comprised of leukocytes (B, NK, and T cells), small cells are predominantly erythrocytes (Ter119+). (C,D) Three distinct peaks can be visualized by ow cytometry. Forthe Scepter cell counter, the size of the debris peak is below the minimum particle diameter for detection.

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esultsMouse splenocytes were analyzed by multicolor owcytometry (Fig. 5). Size-based discrimination con rmed thepresence of two distinct cell populations in all samples.Staining with antibodies speci c for B cells (B220) anderythrocytes (Ter-119) demonstrated that erythrocyteswere restricted to the subset containing smaller cells,while the fraction of large cells was composed primarily of lymphocytes.

Flow-based histograms showed three distinct populationscorresponding to debris, lymphocytes (large cells) anderythrocytes (small cells). Analysis of splenic cell isolatesfrom infected and control mice revealed an overall shift inthe relative cell distribution. Speci cally, infected samplesshowed an increase in % erythrocytes when compared tothe lymphocyte subset (Fig. 5C and Table 2). In contrast,the Scepter cell counter was able to detect only two peaks;

the debris fraction was smaller than the minimum particlediameter for detection. The erythrocytes fraction was alsoat the limits of detection potentially skewing the subsetscount. However, the Scepter cell counters quantitationof splenocyte counts and percentages were in very goodagreement with the values determined by ow cytometry.An assessment of cell concentration values indicated theshift was due to an expansion of splenic erythrocytes whilelymphocyte values remained constant (Table 2). These

ndings were consistent with previously published results5.

Overall ConclusionsEvaluation of immune responsiveness to viral, bacterial,and environmental exposure is an important tool in fordetermining pathogenicity and toxicity. The availabilityof simpli ed methods for identifying activation states

and measuring differences in the relative frequencies(and concentrations) of multiple cell types in samples isvery useful for accelerating research in these elds. .Wehave presented data indicating that the guava easyCytebenchtop ow cytometer and the Scepter cell countercan determine such differences in fresh primary isolatesand cultured samples. While the ow cytometer is themore sophisticated and quantitative tool, the Scepter cell

counter offers rapid, on-line sample quali cation andserves as an adjunct to existing methods. The ability of theScepter device to ensure reproducible cell counts improvesdata quality during experimental setup and downstreamcell-based analyses.

Flow Scepter Concentration (x10 5)

est Sample Erythrocyte Lymphocyte Erythrocyte Lymphocyte Erythrocyte Lymphocyte

Control-1 16 84 27 73 1.86 4.65

Control-2 21 79 29 71 2.61 5.54Control-3 14 86 22 78 2.17 5.78

Infected - 1 42 58 40 60 4.3 5.22

Infected - 2 29 71 30 70 2.82 4.93

Infected - 3 38 62 33 68 3.5 5.84

able 2. Subset frequencies from six splenocyte isolates. Aliquots from each sample were analyzed by ow cytometryand the Scepter cell counter. Cell concentrations were derived on the using the Scepter cell counter.

eferences1. Smith, J et al. The New Scepter 2.0 Cell Counter Enables the

Analysis of a Wider Range of Cell Sizes and Types With HighPrecision. EMD Millipore Cellutions 2011 Vol. 1: p 19-22.

2. Daniels, V. G., Wheater, P. R., & Burkitt, H. G. (1979). FunctionalHistology: A Text and Colour Atlas. Edinburgh: ChurchillLivingstone. ISBN 0-443-01657-7.

3. Prager, E. et al. (2001) Induction of Hyporesponsiveness andImpaired T Lymphocyte Activation by the CD31 Receptor:Ligand Pathway in T-Cells. J. Immunol. 166: 2364-2371.

4. Levine, B. L., et al. (1997). Effects of CD28 Costimulation onLong-term Proliferation of CD4+ T-cells in the Absence of Exogenous Feeder Cells. J. Immunol. 159:5921 5930.

5. MacNamara, K.C., et. al. Diminished Hematopoietic ActivityAssociated with Alterations in Innate and Adaptive Immunityin a Mouse Model of Human Monocytic Ehrlichiosis. Infect.And Immunity. 2009; 77:4061-69.


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Description Quantity Catalog No.

Scepter 2.0 Handheld Automated Cell Counter

with 40 m Scepter Sensors (50 Pack) 1 PHCC20040

with 60 m Scepter Sensors (50 Pack) 1 PHCC20060Includes:

Scepter Cell Counter 1

Downloadable Scepter Software 1

O-Rings 2

Scepter Test Beads 1 PHCCBEADS

Scepter USB Cable 1 PHCCCABLE

Scepter Sensors, 60 m 50 PHCC60050

50 0 PHCC60500

Scepter Sensors, 40 m 50 PHCC40050

50 0 PHCC40500

Universal Power Adapter 1 PHCCP0WER

Scepter O-Ring Kit, includes 2 O-rings and 1 lter cover 1 PHCC0CLIP

Description Quantity Catalog No.

High- hroughput Sampling Instruments

guava easyCyte 8HT Base System 1 0500-4008

guava easyCyte 6HT/2L Base System 1 0500-4007

guava easyCyte 5HT Base System 1 0500-4005

PCA-96 Base System 1 0100-8710

Single Sampling Instruments

easyCyte 5 Base System 1 0500-5005

easyCyte 6-2L Base System 1 0500-5007

easyCyte 8 Base System 1 0500-5008

Guava easyCyte Benchtop Flow Cytometers

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qPCPCR primer pairs were pre-aliquoted in optically clear96-well plates. DNA was ampli ed using a SYBR Green-containing master mix and a fast block protocol. PCRmethod can be used with input DNA ranging from 0.2 to20 ng.

Calculation of elative Copy NumbersCt values obtained for each of the four genes were usedonce to determine mitochondrial DNA copy numbers. Thisis accomplished by averaging the copy numbers calculatedfrom the ND1/BECN1 and the ND6/NEB pairs. The Ct valuesfrom the ND1 gene are subtracted from the BECN1 Ctto obtain Ct1. Likewise; ND6 Ct is subtracted from NEBto obtain Ct2. To calculate copy number, we used theaverage of Set 1 = (ND1/BECN1) and Set 2 = (ND6/NEB)

ratios. To determine the individual ratios from set 1 and set2 the following calculation was used: N = 2Ct where Ct1 = CtNuc1-CtMito1 and Ct2 = CtNuc2-CtMito2.

ho Zero Cell Line143B rho zero cells (143B0; cells devoid of mitochondria)were created by growing 143B cells (human osteosarcomacells, TK-) in their standard media (MEM, 5% FBS,100 g/ml 5-bromo-2-deoxyuridine) in the presence of 50 ng/mL ethidium bromide to achieve 0 status and50 g/mL uridine, which is needed for cell viability oncecells reach glycolytic state. Approximately two months afterstart of ethidium bromide treatment (8 passages), cellsachieved full 0 status and remained stable post removal of ethidium bromide from the media.

Stem Cell Lin

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Cellutions 2011V2 - The Newsletter for Cell Biology Researchers