LifeAct Actin Visualization in Cells

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

2. 2
Mechanisms and Methods of
Molecular Transfer into Living Cells
LifeAct
Protein
LifeAct
Protein
Fuse-It-P
Fuse-It-Beads
Beads
Fuse-It-Beads
Transfer beads and nano-
particles into the cytoplasm
biotin
biotin
biotin
Fuse-It-B
Biotinylate the cell membrane
for versatile use
biotin
biotin
biotin
Fuse-It-B
TRANSLATI
mRNA
Fuse-It-mRNA
mRNA
TRANSLATION
Protein
ds-siRNA
ss-siRNA
RISC
TRAN
Fuse-It-siRNA
siRNA
mRNA
DEGRADATION
PROTEIN
EXPRESSION
Fuse-It-siRNA
Silence your gene of interest
even in sensitive cells
Fuse-It-P
Immediately transfer soluble
proteins into living cells
LifeAct-TagGFP2 Protein
Rapidly visualize F-actin
in living cells

3. 3
Fuse-It-Color
Lipids
Fuse-It-L
rAV-LifeAct
Virus Particle
CAR
TRANSLATION
ssRNAdsDNA
RELEASE
OF RNA
REVERSE
TRAN-
SCRIPTION
STABLE
INTEGRATION
rLV Ubi-LifeAct
Virus Particle
RELEASE
OF DNA
LifeAct AdenoviralVectors
Visualize F-actin in difficult-to-
transfect cells
LifeAct Lentiviral Vectors
Generate stable cell lines for
F-actin visualization
LifeAct
Protein
mRNA
Endosome
ION
pLifeAct
Plasmid
Transfection
Reagent
Endosome
RELEASE
OF DNA
LifeAct
Protein
NSCRIPTION
Fuse-It-mRNA
Transfer mRNA fast and
directly into the cytoplasm
LifeAct Plasmids
Get brilliant F-actin staining
in living cells
Fuse-It-L
Incorporate lipids into
the plasma membrane
Fuse-It-Color
Label the plasma membrane
with various dyes
Nucleus
3

4. 4
The Fuse-It Technology
Endocytotic
Liposome
Endosome
ENDOSOMAL
RELEASE
Fusogenic
Liposome
Membrane Fusion Lipofection
LYSOSOMAL
DEGRADATION
Lysosome
ENDOCYTOSIS
FUSION
Liposomal
Carrier
Lipoplex
IMMEDIATE RELEASE
INTO 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.
Csiszár, 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

5. 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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p. 7
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p. 9
p. 9
p. 9
Fuse-It-L
A reagent for lipid incorporation
into the plasma membrane
of living cells for mutational
investigations
p. 7
in cooperation with
5
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
p. 10
p. 10
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p. 10
* in cooperation with

6. 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.
0
10
20
30
40
50
60
70
80
90
100
Geneexpression/Viability[%]
Gene expression Viability
ds-siRNA
ss-siRNA
RISC
Fuse-It-siRNA
siRNA
mRNA
DEGRADATION
PROTEIN
EXPRESSION
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
6
Fuse-It-siRNA

7. Competitor
Fuse-It-mRNA
mRNA
TRANSLATION
Protein
• Efficiently and rapidly transfer mRNA 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
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
7

8. 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
of artificial overexpression
• 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.
Instant Protein Activity
Bright staining of the actin cytoskeleton of myofibro-
blasts already 5 minutes after fusion with Fuse-It-P
and LifeAct Protein.
Fuse-It-P
Immediate Transfer of Soluble Proteins
Protein
Protein
Fuse-It-P
8
Phase
Contrast
1 min
Infrared
Control Dye
3 min
R-Phycoerythrin 5 min

9. 9
Hersch, N., et al., Biotin-conjugated
fusogenic liposomes for high-
quality cell purification. J Biomater
Appl, 30(6), 846–856 (2016).
Fuse-It-Color
• Efficiently label the plasma membranes of
living 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-Beads
Transfer 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

10. www. .com
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:
LifeAct
Visualization 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 expressing
LifeAct-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 | order@ibidi.de
ibidi USA, Inc. | 844.276.6363 | ibidiusa@ibidi.com
©ibidiGmbH,V 1.0,2017 / 10