Fuse-It Membrane Fusion

Transcription

1 Fuse-It Membrane Fusion Next Generation Transfection Including: LifeAct Actin Visualization in Living Cells

2 Mechanisms and Methods of Molecular Transfer into Living Cells Fuse-It- Fuse-It-siRNA Silence your gene of interest even in sensitive cells sirna Fuse-It-siRNA TRANSLATION Fuse-It-P ds-sirna Immediately transfer soluble proteins into living cells ss-sirna TRANSLATI RISC Fuse-It-P TRAN DEGRADATION PROTEIN EXPRESSION biot in in ot bi biotin Fuse-It-Beads Beads biotin tin Transfer beads and nanoparticles into the cytoplasm biotin Fuse-It-Beads bio Fuse-It-B Fuse-It-B Biotinylate the cell membrane for versatile use 2

3 Fuse-It- Transfer fast and directly into the cytoplasm LifeAct Plasmids Get brilliant F-actin staining in living cells plifeact Plasmid Transfection Reagent LifeAct LifeAct Adenoviral Vectors Visualize F-actin in difficult-totransfect cells CAR Endosome ION rav-lifeact Virus Particle Endosome RELEASE OF DNA RELEASE OF DNA NSCRIPTION STABLE INTEGRATION Nucleus TRANSLATION LifeAct LifeAct Lentiviral Vectors RELEASE OF RNA Generate stable cell lines for F-actin visualization dsdna REVERSE ssrna TRANSCRIPTION rlv Ubi-LifeAct Virus Particle Lipids Fuse-It-Color Fuse-It-Color Label the plasma membrane with various dyes Fuse-It-L Incorporate lipids into the plasma membrane Fuse-It-L 3

4 The Fuse-It Technology Fusogenic Liposome Endocytotic Liposome Liposomal Carrier Lipoplex Membrane Fusion FUSION Lipofection ENDOCYTOSIS IMMEDIATE RELEASE INTO THE CYTOPLASM Endosome Fuse-It Membrane Fusion Is Superior to Lipofection ENDOSOMAL RELEASE 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. LYSOSOMAL DEGRADATION Lysosome Cardiomyocytes T cells Macrophages Endothelial cells Supreme biocompatibility: In contrast to classical lipofection reagents, the Fuse-It reagents are non-toxic. Even sensitive and difficult-totransfect cells, such as primary neurons, keratinocytes, and stem cells, retain high viability with maximized fusion efficiency. Keratinocytes Neurons Stem cells Csisza r, A., et al., Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification. Bioconjugate Chem, 21(3), (2010). 4

5 Product Overview Fuse-It Membrane Fusion in cooperation with Fuse-It-siRNA p. 6 A fusion-mediated sirna transfection reagent for rapid and efficient gene silencing with maximized biocompatibility Product Overview LifeAct Actin Visualization * in cooperation with Fuse-It- p. 7 A reagent for fusion-mediated transfection for immediate analysis of living cells without side effects LifeAct Plasmids p. 10 Fuse-It-P p. 8 A range of plasmids for transient or stable transfections of various cell types; useful for brilliant visualization of F-actin A fusion reagent for immediate delivery of proteins into living cells for investigations without artificial overexpression LifeAct Adenoviral Vectors * p. 10 Ready-to-use adenoviral vectors for efficient F-actin transduction, especially suitable for studies in difficult-to-transfect cells Fuse-It-Color p. 9 A fusion-based dye for quick and efficient labeling of living cells while retaining complete cell function and viability LifeAct Lentiviral Vectors * p. 10 Lentiviral vectors for easy generation of stable LifeActexpressing cell lines with unrestricted actin functionality Fuse-It-Beads p. 9 A reagent for fusion-mediated incorporation of beads and nanoparticles into the cytoplasm for various experimental purposes LifeAct-TagGFP2 p. 10 A recombinant protein for remarkably fast staining and immediate functional analysis of F-actin in living and fixed cells Fuse-It-B p. 9 A fusion reagent for biotinylation of the cell membrane for applications taking advantage of the biotin-avidin affinity LifeAct Stable Cell Line p. 10 A stable LifeAct-expressing human fibro-sarcoma cell line with full actin functionality for direct use in cell-based assays Fuse-It-L p. 7 A reagent for lipid incorporation into the plasma membrane of living cells for mutational investigations 5

6 Fuse-It-siRNA Improved sirna Transfection Silence your gene of interest with maximized biocompatibility sirna 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. Fuse-It-siRNA ds-sirna ss-sirna RISC DEGRADATION Control Gene expression / Viability [%] PROTEIN EXPRESSION Competitor Gene expression Viability Fuse-It-siRNA 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 knockdown and retained viability, even after multiple Fuse-It-siRNA transfers in primary human keratinocytes. Cat. No. Description Fuse-It-siRNA, infrared fluorescent: 6 mm, 2 x 150 µl Fuse-It-siRNA, infrared fluorescent: 6 mm, 2 x 300 μl 6

7 Fuse-It- The Shortcut for Expression Efficiently and rapidly transfer for protein analysis 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 Fuse-It- Maximized biocompatibility: Due to low cytotoxicity, Fuse-It- is especially suitable for sensitive primary and non-dividing cells. Direct analysis: 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. TRANSLATION HUVEC ipsc Competitor Cortical Neurons Validated Efficiency in Various Cell Lines GFP expression in indicated cell types after GFP- transfection with Fuse-It-. Check out your cell type of interest at Fuse-It- Health and Viability Sixteen hours after GFP- transfer with Fuse-It-, the viability of nhek cells is retained, whereas significant cell death occurs after using classical lipoplexbased methods. Cat. No. Description Fuse-It-, infrared fluorescent: 6 mm, 2 x 150 µl Fuse-It-, infrared fluorescent: 6 mm, 2 x 300 μl 7

8 Fuse-It-P Immediate Transfer of Soluble s Deliver your protein of interest directly into living cells without the interfering effects of artificial overexpression Perform functional imaging, such as speckle analysis, FRAP, or single molecule analysis Fuse-It-P Controlled quantity: Fuse-It-P is optimized for the efficient transfer of low to intermediate amounts of water-soluble proteins. Instant activity: s are freed directly into the cytoplasm within 1 20 minutes. Efficient transfer: No lysosomal degradation can occur, which is in contrast to endosomal uptakedepending protein transfection methods. Versatile application: Use the most suitable buffer for your protein. 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), L (2015). Kube, S., et al., Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular s. Langmuir, 33(4), (2017). Phase Contrast Infrared Control Dye R-Phycoerythrin Efficiency and Easy Visualization CHO-K1 cells directly after fusion with Fuse-It-P and R-Phycoerythrin. Note: When using Fuse-It-P, the amount of transfected protein is much less in comparison to plasmid DNA or 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. Cat. No. Description Fuse-It-P, infrared fluorescent: lyophilized, for 100 µl solution (3 mm) Fuse-It-P, infrared fluorescent: lyophilized, for 4 x 25 µl solution (3 mm) Fuse-It-P, infrared fluorescent: lyophilized, for 400 µl solution (3 mm) Fuse-It-P, infrared fluorescent: lyophilized, for 4 x 100 µl solution (3 mm) 8

9 Be Versatile More Fuse-It Products for Your Applications Fuse-It-Color Efficiently label the plasma membranes of living cells while completely retaining cell function and viability Fuse-It-B Biotinylate the surface of living cells biotin 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. Hersch, N., et al., Biotin-conjugated fusogenic liposomes for highquality cell purification. J Biomater Appl, 30(6), (2016). Fuse-It green CHO cells fused with the indicated Fuse-It- Color dyes for 1 minute. Fuse-It-Beads Transfer beads and nanoparticles into the cytoplasm beads Fuse-It red Fuse-It dred Moch, M., et al., Effects of Plectin Depletion on Keratin Network Dynamics and Organization. PLOS ONE, 11(3), e (2016). Fuse-It IR Fuse-It-L Incorporate lipids into the plasma membrane lipid Ma, Y., et al., A FRET sensor enables quantitative measurements of membrane charges in live cells. Nat Biotechnol, 35(4), (2017). Find more detailed information at: Cat. No. Description Fuse-It-Beads, infrared fluorescent: 3 mm, 100 μl Fuse-It green, green fluorescent: 3 mm, 100 μl Fuse-It red, red fluorescent: 3 mm, 100 μl Fuse-It dred, dark red fluorescent: 3 mm, 100 μl Fuse-It IR, infrared fluorescent: 3 mm, 100 μl Fuse-It-B, green fluorescent: 3 mm, 100 μl Fuse-It-B, infrared fluorescent: 3 mm, 100 μl Fuse-It-L, infrared fluorescent: lyophilized, for 100 μl solution (3 mm) 9

10 LifeAct Visualization of F-Actin in Living Cells Brilliantly visualize F-actin with unrestricted functionality no interference with cytoskeletal 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. Z-stack of HT-1080 LifeAct-TagGFP2 cells in a 3D hydrogel environment. 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 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 longterm expression into dividing and non-dividing cells, both in vitro and in vivo. LifeAct The recombinant LifeAct is especially useful for very fast staining of F-actin in living cells. In fixed cells, it represents the non-toxic alternative to phalloidin. 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. Original Publications: Riedl J., et al., Lifeact a versatile marker for the visualization of F-actin. Nature Methods 5, (2008). Riedl J., et al., Lifeact-mice for studying F-actin dynamics. Nature Methods 7, (2010). Find the ideal LifeAct product for your application: ibidi GmbH, V 1.1, 2018 / 05 Cat. No. Description p CMV -LifeAct-TagGFP2: plasmid, lyophilized, 20 µg p CMV -LifeAct-TagRFP: plasmid, lyophilized, 20 µg p CAG -LifeAct-TagGFP2: plasmid, lyophilized, 20 µg p CAG -LifeAct-TagRFP: plasmid, lyophilized, 20 µg LifeAct-TagGFP2: lyophilized, 1 x 100 µg LifeAct-TagGFP2: lyophilized, 4 x 100 µg rav CMV -LifeAct-TagGFP2: adenoviral vector, 1x10 10 IU/ml, 1x10 9 IU rav CMV -LifeAct-TagRFP: adenoviral vector, 1x10 10 IU/ml, 1x10 9 IU rlv Ubi -LifeAct-TagGFP2: lentiviral vector, 1x10 7 TU/ml, 100 μl rlv Ubi -LifeAct-TagRFP: lentiviral vector, 1x10 7 TU/ml, 100 μl HT-1080 LifeAct-TagGFP2: HT-1080 cells expressing LifeAct-TagGFP2, 5x10 5 cells/vial 10 ibidi GmbH / order@ibidi.de ibidi USA, Inc ibidiusa@ibidi.com