Fuse-It Membrane FusionNext Generation Transfection

Including:

LifeAct—Actin Visualization in Living Cells

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Mechanisms and Methods of Molecular Transfer into Living Cells

Protein

ProteinFuse-It-P

Fuse-It-Beads

Beads

Fuse-It-BeadsTransfer beads and nano-particles into the cytoplasm

biotin

biotin

biotin

Fuse-It-BBiotinylate the cell membrane for versatile use

biot

in

biotin

biotin

Fuse-It-B

Fuse-It-Color

Lipids

Fuse-It-L

rAV-LifeActVirus Particle

CAR

TRANSLATION

ssRNAdsDNA

RELEASEOF RNA

REVERSETRAN-

SCRIPTION

STABLEINTEGRATION

rLV Ubi-LifeActVirus Particle

RELEASEOF DNA

LifeAct Adenoviral VectorsVisualize F-actin in difficult-to-transfect cells

LifeAct Lentiviral VectorsGenerate stable cell lines for F-actin visualization

LifeActProtein

mRNA

Endosome

TRANSLATION

mRNA

pLifeActPlasmid

TransfectionReagent

Endosome

RELEASEOF DNA

LifeActProtein

Fuse-It-mRNAmRNA

TRANSLATION

Protein

ds-siRNA

ss-siRNA

RISC

TRANSCRIPTION

Fuse-It-siRNA

siRNA

mRNA

DEGRADATION

PROTEINEXPRESSION

Fuse-It-siRNASilence your gene of interest even in sensitive cells

Fuse-It-mRNATransfer mRNA fast and directly into the cytoplasm

LifeAct PlasmidsGet brilliant F-actin staining in living cells

Fuse-It-LIncorporate lipids into the plasma membrane

Fuse-It-PImmediately transfer soluble proteins into living cells

Fuse-It-ColorLabel the plasma membrane with various dyes

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Protein

ProteinFuse-It-P

Fuse-It-Beads

Beads

Fuse-It-BeadsTransfer beads and nano-particles into the cytoplasm

biotin

biotin

biotin

Fuse-It-BBiotinylate the cell membrane for versatile use

biot

in

biotinbiotin

Fuse-It-B

Fuse-It-Color

Lipids

Fuse-It-L

rAV-LifeActVirus Particle

CAR

TRANSLATION

ssRNAdsDNA

RELEASEOF RNA

REVERSETRAN-

SCRIPTION

STABLEINTEGRATION

rLV Ubi-LifeActVirus Particle

RELEASEOF DNA

LifeAct Adenoviral VectorsVisualize F-actin in difficult-to-transfect cells

LifeAct Lentiviral VectorsGenerate stable cell lines for F-actin visualization

LifeActProtein

mRNA

Endosome

TRANSLATION

mRNA

pLifeActPlasmid

TransfectionReagent

Endosome

RELEASEOF DNA

LifeActProtein

Fuse-It-mRNAmRNA

TRANSLATION

Protein

ds-siRNA

ss-siRNA

RISC

TRANSCRIPTION

Fuse-It-siRNA

siRNA

mRNA

DEGRADATION

PROTEINEXPRESSION

Fuse-It-siRNASilence your gene of interest even in sensitive cells

Fuse-It-mRNATransfer mRNA fast and directly into the cytoplasm

LifeAct PlasmidsGet brilliant F-actin staining in living cells

Fuse-It-LIncorporate lipids into the plasma membrane

Fuse-It-PImmediately transfer soluble proteins into living cells

Fuse-It-ColorLabel the plasma membrane with various dyes

Nucleus

3

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The Fuse-It Technology

EndocytoticLiposome

Endosome

ENDOSOMALRELEASE

FusogenicLiposome

Membrane Fusion Lipofection

LYSOSOMALDEGRADATION

Lysosome

ENDOCYTOSIS

FUSION

LiposomalCarrier

Lipoplex

IMMEDIATE RELEASEINTO THE CYTOPLASM

Supreme biocompatibility: In contrast to classical lipofection reagents, the Fuse-It reagents are non-toxic. Even sensitive and difficult-to- transfect cells, such as primary neurons, keratino- cytes, and stem cells, retain high viability with maximized fusion efficiency.

Fuse-It Membrane Fusion Is Superior to Lipofection

Basic principle: The Fuse-It liposomal carrier simply fuses with the cell membrane, then releases the included molecule of interest directly into the cytoplasm. This results in immediate and efficient transfer without processes such as endocytosis, lysosomal degradation, and mitosis.

Csiszar, A., et al., Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification. Bioconjugate Chem, 21(3), 537–543 (2010).

Neurons

Endothelial cells

Cardiomyocytes Macrophages

T cells

Keratinocytes Stem cells

Fuse-It-mRNA

A reagent for fusion-mediated mRNA transfection for imme- diate analysis of living cells without side effects

Fuse-It-siRNA

A fusion-mediated siRNA trans- fection reagent for rapid and efficient gene silencing with maximized biocompatibility

Fuse-It-Color

A fusion-based dye for quick and efficient labeling of living cells while retaining complete cell function and viability

Fuse-It-P

A fusion reagent for immediate delivery of proteins into living cells for investigations without artificial overexpression

Fuse-It-Beads

A reagent for fusion-mediated incorporation of beads and nano- particles into the cytoplasm for various experimental purposes

Fuse-It-B

A fusion reagent for biotinylation of the cell membrane for appli-cations taking advantage of the biotin-avidin affinity

Product Overview

Fuse-It Membrane Fusion

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Fuse-It-L

A reagent for lipid incorporation into the plasma membrane of living cells for mutational investigations

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Product Overview

LifeAct Actin Visualization

LifeAct Plasmids

A range of plasmids for tran-sient or stable transfections of various cell types; useful for brilliant visualization of F-actin

LifeAct-TagGFP2 Protein A recombinant protein for re- markably fast staining and im-mediate functional analysis of F-actin in living and fixed cells

LifeAct Stable Cell Line A stable LifeAct-expressing human fibro-sarcoma cell line with full actin functionality for direct use in cell-based assays

LifeAct Adeno- viral Vectors * Ready-to-use adenoviral vectors for efficient F-actin transduction, especially suitable for studies in difficult-to-transfect cells

LifeAct Lentiviral Vectors *

Lentiviral vectors for easy generation of stable LifeAct-expressing cell lines with unrestricted actin functionality

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Control

Competitor

• Silence your gene of interest with maximized biocompatibility

• Concentrate on the real knockdown phenotype, instead of side effects caused by the transfection reagent

Highest viability: Fuse-It-siRNA shows extremely low cytotoxicity, thereby retaining cellular function, also in primary and non-dividing cells, such as keratinocytes.

Efficient delivery: siRNA is transferred directly into the cytoplasm without any interference by endocytosis or lysosomal degradation.

Less time: Successful siRNA transfer is achieved within only 5–20 minutes.

High Efficiency and Viability

Fuse-It-siRNA provides more efficient GFP knockdown and retains healthy morphology in CHO-K1 cells, in contrast to classical siRNA transfection reagents (competitor).

Maximized efficiency of α-Catenin knock-down and retained viability, even after multiple Fuse-It-siRNA transfers in primary human keratinocytes.

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Gene expression Viability

ds-siRNA

ss-siRNA

RISC

TRANSCRIPTION

Fuse-It-siRNA

siRNA

mRNA

DEGRADATION

PROTEINEXPRESSION

Cat. No. Description

60510 Fuse-It-siRNA, infrared fluorescent: 6 mM, 2 x 150 µl

60511 Fuse-It-siRNA, infrared fluorescent: 6 mM, 2 x 300 µl

Fuse-It-siRNA Improved siRNA Transfection

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Fuse-It-siRNA

Competitor

Fuse-It-mRNAmRNA

TRANSLATION

Protein

• EfficientlyandrapidlytransfermRNAforproteinanalysis—be protected against side effects and loss of time by plasmid DNA integration

• Directly transfect sensitive primary and non-dividing cells such as neurons, endothelial cells, and stem cells

Maximized biocompatibility: Due to low cytotoxicity, Fuse-It-mRNA is especially suitable for sensitive primary and non-dividing cells.

Direct analysis: mRNA transfer occurs fast and is completed within only 5–20 minutes.

High efficiency: Interfering processes, such as endocytosis, lysosomal degradation, or gene transfer to the nucleus, are omitted.

Simple application: No genetically modified organisms (GMOs) are generated and no Biosafety Laboratory Level 1 or 2 is needed.

Validated Efficiency in Various Cell Lines

GFP expression in indicated cell types after GFP-mRNA transfection with Fuse-It-mRNA. Check out your cell type of interest at www.ibidi.com.

Health and Viability

Sixteen hours after GFP-mRNA transfer with Fuse-It-mRNA, the viability of nHEK cells is retained, whereas significant cell death occurs after using classical lipoplex-based methods.

Fuse-It-mRNA

HUVEC

Cortical Neurons

iPSC

Cat. No. Description

60500 Fuse-It-mRNA, infrared fluorescent: 6 mM, 2 x 150 µl

60501 Fuse-It-mRNA, infrared fluorescent: 6 mM, 2 x 300 µl

Fuse-It-mRNA The Shortcut for Protein Expression

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Burgstaller, G., et al., Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue. Am J Physiol Lung Cell Mol Physiol, 309(4), L323 –32 (2015).

Kube, S., et al., Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins. Langmuir, 33(4), 1051–1059 (2017).

• Deliver your protein of interest directly into living cells without the interfering effects ofartificialoverexpression

• Perform functional imaging, such as speckle analysis, FRAP, or single molecule analysis

Controlled quantity: Fuse-It-P is optimized for the efficient transfer of low to intermediate amounts of water-soluble proteins.

Instant activity: Proteins are freed directly into the cytoplasm within 1–20 minutes.

Efficient transfer: No lysosomal degradation can occur, which is in contrast to endosomal uptake-depending protein transfection methods.

Versatile application: Use the most suitable buffer for your protein.

Cat. No. Description

60220 Fuse-It-P, infrared fluorescent: lyophilized, for 100 µl solution (3 mM)

60221 Fuse-It-P, infrared fluorescent: lyophilized, for 4 x 25 µl solution (3 mM)

60222 Fuse-It-P, infrared fluorescent: lyophilized, for 400 µl solution (3 mM)

60223 Fuse-It-P, infrared fluorescent: lyophilized, for 4 x 100 µl solution (3 mM)

Efficiency and Easy Visualization

CHO-K1 cells directly after fusion with Fuse-It-P and R-Phycoerythrin.

Fuse-It-P Immediate Transfer of Soluble Proteins

Protein

ProteinFuse-It-P

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Phase Contrast

Infrared Control Dye

R-Phycoerythrin

Note: When using Fuse-It-P, the amount of transfected protein is much less in comparison to plasmid DNA or mRNA transfection. Since there is no amplification of the delivered proteins, the fluorescence signal of a transfected reporter protein, such as GFP, is generally weak. What seems to be a disadvantage for fluorescence microscopy is a benefit for protein functionality: when using Fuse-It-P, the proteins are delivered at a physiological level without artificial overexpression, keeping their natural functionality inside the living cell.

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Hersch, N., et al., Biotin-conjugated fusogenic liposomes for high- quality cell purification. J Biomater Appl, 30(6), 846–856 (2016).

Fuse-It-Color

• Efficientlylabeltheplasmamembranesofliving cells while completely retaining cell function and viability

Immediate analysis: Perform live-cell microscopy, co-culture experiments, FACS, and more.

Various dyes: Fuse-It-Color is provided in green, red, dark red, and infrared.

CHO cells fused with the indicated Fuse-It-Color dyes for 1 minute.

Fuse-It green

Fuse-It red

Fuse-It dred

Fuse-It IR

Find more detailed information at:

www.ibidi.com

Be Versatile More Fuse-It Products for Your Applications

Moch, M., et al., Effects of Plectin Depletion on Keratin Network Dynamics and Organization. PLOS ONE, 11(3), e0149106 (2016).

Fuse-It-BeadsTransfer beads and nanoparticles into the cytoplasm beads

Fuse-It-B

Biotinylate the surface of living cells

Cat. No. Description

60200 Fuse-It green, green fluorescent: 3 mM, 100 µl

60202 Fuse-It red, red fluorescent: 3 mM, 100 µl

60204 Fuse-It dred, dark red fluorescent: 3 mM, 100 µl

60206 Fuse-It IR, infrared fluorescent: 3 mM, 100 µl

60420 Fuse-It-Beads, infrared fluorescent: 3 mM, 100 µl

60320 Fuse-It-B, green fluorescent: 3 mM, 100 µl

60322 Fuse-It-B, infrared fluorescent: 3 mM, 100 µl

60210 Fuse-It-L, infrared fluorescent: lyophilized, for 100 µl solution (3 mM)

biotin

Ma, Y., et al., A FRET sensor enables quantitative measure-ments of membrane charges in live cells. Nat Biotechnol, 35(4), 363–370 (2017).

Fuse-It-L

Incorporate lipids into the plasma membrane

lipid

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Original Publications:

Riedl J., et al., Lifeact – a versatile marker for the visualization of F-actin. Nature Methods 5, 605–607 (2008).

Riedl J., et al., Lifeact-mice for studying F-actin dynamics. Nature Methods 7, 168–169 (2010).

• Brilliantly visualize F-actin with unrestricted functionality—no interference with cyto-skeletal dynamics in vitro and in vivo

LifeAct, a 17-amino acid peptide, is derived from a protein found in Saccharomyces cerevisiae. It stains filamentous actin (F-actin) structures in living or fixed eukaryotic cells and tissues with excellent signal-to-noise ratio.

Cat. No. Description

60101 p CMV-LifeAct-TagGFP2: plasmid, lyophilized, 20 µg

60102 p CMV-LifeAct-TagRFP: plasmid, lyophilized, 20 µg

60106 p CAG-LifeAct-TagGFP2: plasmid, lyophilized, 20 µg

60107 p CAG-LifeAct-TagRFP: plasmid, lyophilized, 20 µg

60112 Protein LifeAct-TagGFP2: lyophilized, 1 x 100 µg

60113 Protein LifeAct-TagGFP2: lyophilized, 4 x 100 µg

Find the ideal LifeAct product for your application:

LifeActVisualization of F-Actin in Living Cells

60121 rAV CMV-LifeAct-TagGFP2: adenoviral vector,1x1010 IU/ml, 1x109 IU

60122 rAV CMV-LifeAct-TagRFP: adenoviral vector,1x1010 IU/ml, 1x109 IU

60141 rLV Ubi-LifeAct-TagGFP2: lentiviral vector,1x107 TU/ml, 100 µl

60142 rLV Ubi-LifeAct-TagRFP: lentiviral vector,1x107 TU/ml, 100 µl

40101 HT-1080 LifeAct-TagGFP2: HT-1080 cells expressingLifeAct-TagGFP2, 5x105 cells/vial

LifeAct Plasmid

After transient or stable transfection of cells with the LifeAct plasmid, F-actin can be brilliantly visualized using the fluorescence markers GFP2 or RFP. Please note: The LifeAct Plasmid is not compatible with the available Fuse-It products.

LifeAct Protein

The recombinant LifeAct Protein is especially useful for very fast staining of F-actin in living cells. In fixed cells, it represents the non-toxic alternative to phalloidin.

LifeAct Adenoviral and Lentiviral Vectors

The LifeAct viral vectors are especially suited for difficult-to-transfect cells and for the generation of stable cell lines. They mediate efficient transduction, integration, and long-term expression into dividing and non-dividing cells, both in vitro and in vivo.

Stable LifeAct Expressing Cell Line

The stable human fibrosarcoma cell line HT-1080 LifeAct-TagGFP2 allows perfect visualization of highly dynamic F-actin, which is ideal for studies on migration, chemotaxis, and wound healing.

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Z-stack of HT-1080 LifeAct-TagGFP2 cells in a 3D hydrogel environment.

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ibidi USA, Inc. | 844.276.6363 | ibidiusa@ibidi.com

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