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FAQ: LifeAct Actin Visualization


What is the difference between using the CMV or CAG promoters in the LifeAct plasmids and adenoviral vectors?

The promoter that you choose, either CMV (original source: cytomegalovirus) or CAG (original source: chicken, a modified β-actin promoter), will depend on the type of cell that you are using. For many standard cell lines, the CMV promoter is the best choice, because it leads to the strong expression of LifeAct. However, when using sensitive cells, such as neuronal or embryonic stem cells, we almost always recommended using the CAG promoter. It does not have a viral origin, so an interference with cellular defense mechanisms (“silencing mechanisms”) is less likely.


Which transfection method is the best for working with LifeAct plasmids?

Basically, every available transfection method will also work with our LifeAct plasmids. If you already have experience with a specific cell type, we recommend using the same method. If you are new to this field, please refer to published literature, or try different methods (e.g., lipofection, electroporation, or nucleofection) to find the appropriate transfection method for your cells.


Which transfection method works best for neurons, when working with LifeAct?

If you have already experience with a specific cell-type, we recommend using the same method, when working with LifeAct. If you are new to this field, please refer to published literature, or try different methods (e.g., lipofection, electroporation, or nucleofection) to find the appropriate one for your cells. In our own experiments with primary hippocampal neurons, we used nucleofection to transfer the DNA into the cells.


Which transfection method works best for monocytes, when working with LifeAct?

If you have already experience with a specific cell type, we recommend using the same method, when working with LifeAct. If you are new to this field, please refer to published literature, or try different methods (e.g., lipofection, electroporation, or nucleofection) to find the appropriate one for your cells. In our own experiments with primary, mature dendritic cells, we used nucleofection to transfer the DNA into the cells.


With your LifeAct products, what is the transfection rate into cells?

This strongly depends on the cell type, and the transfection method you are using. In general, we get higher transfection rates with our LifeAct plasmids than, for example, with Actin-GFP plasmids. To find the optimal result for your specific cell-type, we recommend testing different procedures and reagents (e.g., ibidi's Torpedo DNA).


Have you also tried to transfect tonsilar B cells with LifeAct?

Up to now, we have not worked with splenic naïve B cells. However, there is a publication that demonstrates the successful application of LifeAct in primary B cells:

The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor, Treanor et al., Immunity 32, 2010.


LifeAct has a low binding affinity, but it perfectly overlies the F-Actin. How is its binding affinity to G-Actin?

LifeAct very specifically stains F-Actin in eukaryotic cells, even though it shows a low binding affinity in biochemical assays. The big advantage to this is that there is no influence on the actin kinetics in the living cells. It is a great advantage when comparing LifeAct with Actin-GFP.

In biochemical assays, we also found that LifeAct binds to G-Actin with a low affinity. In in vivo experiments, we see a very specific F-Actin staining, with a high signal-to-noise ratio. So, it is not very likely that LifeAct binds G-Actin in a cellular environment, presumably because G-Actin is rarely found in cells without binding cofactors. 


How can you distinguish lamellopodia and filopodia?

Lamellipodia are sheet-like protrusions of cells that are, for example, seen in migrating lymphocytes. They consist of a gel-like network of actin filaments that are highly dynamic.

Filopodia are spike-like protrusions at the edges of cells that are, for example, seen in neurons or fibroblasts. They consist of thick, parallel bundles of actin filaments.

Filopodia in a HT-1080 cell, transiently transfected with a LifeAct plasmid
Lamellipodia in a mouse primary dendritic cell, nucleofected with a LifeAct plasmid

Was LifeAct also tested with apicomplexan organisms?

Yes, LifeAct was successfully tested in plasmodium.


Did you already test LifeAct in primary cultured cells?

Yes, we tested LifeAct in primary hippocampal neurons and primary dendritic cells. There are also publications showing applications in primary macrophages, keratinocytes, or B-cells.


Have you tried LifeAct for mesenchymal stem cells?

We did not investigate hMSCs in our laboratory, however there is a publication showing the application of LifeAct in these cells:

Deformation of stem cell nuclei by nanotopographical cues, Chalut KJ et al., SOFT MATTER (6) 8, 1675-1681, 2010.


Did you try LifeAct with osteoblasts?

We did not investigate osteoblasts, however there is a publication showing the application of LifeAct in chondrocytes:

Profilin 1 is required for abscission during late cytokinesis of chondrocytes, Bottcher RT et al. EMBO JOURNAL 28(8), 1157-1169, 2009.


Which neurons have you tested with LifeAct?

We tested primary hippocampal neurons from rats and mice with LifeAct. They were prepared and then transfected with pCMV LifeAct-TagGFP2 on the next day. After being cultured for several days, the microscopic images were acquired. There are additional publications where, for example, motor neurons were analyzed. Here is one such publication:

Fallini et al. Molecular Neurodegeneration 2010, 5:17


When using LifeAct, have you found staining of the growth cones in neurons?

When using LifeAct, we observed a very bright and specific F-Actin staining in the growth cones of polarizing neurons with high dynamics. That staining is typical for this structure.


Has the LifeAct F-Actin stain already been used with endothelial cells?

Yes, we used our recombinant adenovirus rAV-LifeAct for the transduction of HUVECs, and achieved very good results with this approach.


Is it known which specific LifeAct domains are bound in actin?

To date, we do not know where LifeAct binds to Actin.


Has anyone done automatic image analysis of movies generated with LifeAct-expressing cells?

As far as we know, there is no publication showing automatic image analysis of LifeAct-expressing cells.


How long is LifeAct stable in living cells, and also in fixed samples?

For living cells, it depends strongly on the cell-type you are using. For transient transfections, LifeAct can be expressed for 3 to 5 days, depending on the division rate and the original amount of DNA that was introduced. However, with our LifeAct plasmids, it is also possible to generate stable cell lines, if long-term expression is needed. ibidi also offers customized, specific generation of those stable cell lines. For more information, please send an email to info@ibidi.com.

There is also the possibility to fix cells after transfection, which will then retain the fluorescence signal for further analysis. In the future, ibidi will be providing a LifeAct peptide for application in immunofluorescence, or biochemical assays.  


How does LifeAct compare with competing reagents, such as Actin-FP?

Actin coupled to different fluorescent proteins (Actin-FP), and also used for live-cell imaging, is available from several companies. ibidi, and others, have demonstrated that Actin-FP is less functional than the related, wild-type protein. Plus, it interferes with actin kinetics in sensitive, cellular processes. For cytoskeletal analysis, LifeAct is the best option, as it does not interfere with the cellular dynamics.


Is LifeAct like an expression system? Does it generate fusion proteins?

At the moment, ibidi provides genetically encoded fusion proteins of LifeAct, which are either coupled to TagGFP2, or TagRFP. These versions are available as plasmids, or packaged in adenoviral vectors.


Could the transfection with LifeAct be combined with other physical forces, such as electroporation or sonoporation?

If your experimental approach requires the combination of transfection with other physical forces, combining them to LifeAct is possible, in theory. Additionally, if you already have experience working with such a system, it should also work with our LifeAct plasmids.


Is there any selection marker that can be used for the selection of stable clones, when LifeAct is transduced into cells through adenovirus?

Transduction of recombinant adenoviruses does not lead to random integration into the genome of cells, and therefore it is not possible to generate stable cell lines with this system.

If you are interested in generating or using stable cell lines, ibidi also offers a specific generation of those lines, customized to your needs. For more information, please send an email to info@ibidi.com.


After transfection with LifeAct, how long does the incubation time need to be, before further studies can be made?

In general, many cells already show strong expression of LifeAct fusion proteins, 24 hours after transfection.


Do you have any plans to make an embryonic stem (ES) cell line, or a transgenic mouse expressing LifeAct-GFP?

At the moment, ibidi is not planning to establish a stably expressing ES cell line. However, we are currently generating a transgenic mouse line expressing LifeAct-TagGFP2, which will be made available to our customers, at a later date.