The Effects of Membrane Physical Properties on the Fusion of ...
Fuse-It Membrane Fusion - ibidi · 2019-03-14 · Fuse-It Membrane Fusion Is Superior to...
Transcript of Fuse-It Membrane Fusion - ibidi · 2019-03-14 · Fuse-It Membrane Fusion Is Superior to...
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
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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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Gen
e ex
pres
sion
/ Vi
abilit
y [%
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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.
ibidi GmbH | +49 89 / 520 46 17 - 0 | [email protected]
ibidi USA, Inc. | 844.276.6363 | [email protected]
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