Does anybody know a method to differentiate between cells before and after cell division? We plan a migration assay and therefore we plan the following project:
1. First we seed out HUVEC cells on a slide
2. Treatment of these cells
3. Check the migration of these cells after 24 hours to a part of the slide where cells were at the beginning
So the problem is that in this 24 hours the cells divide and so it is difficult to know: Are the old cells migrated or are the "migrated" cells only new cells after division?
Any staining ideas etc.?
I was thinking the same thing as Sneha, CFSE staining, the read-out for this is flow cytometry, so perhaps you would need to seed much more cells to get an aliquot for flow cytometry or make duplicate slides.
Membrane dyes are one feasible option, provided they and the UV illumination during fluorescent video microscopy do not affect the behavior of your HUVECs. Another technology that might be useful is holographic imaging, which is non-invasive and less damaging than fluorescent light. See http://www.phiab.se/ for more information. While it is does not explicitly distinguish between cells that have vs. have not divided, it enables very effective single cell tracking, so you can determine daughter cells by simply watching a time-lapse image.
My idea is to infect cells with baculoviruses which introduce the fluorophore into the choosen organellum, eg. ER. In my experiments only dauther cells will give You the light, while cells infected at t=0 should not. This method is however not for such purpose, but it could work, basing on my own observations.
You might try BrdU (bromodeoxyuridine )incorporation followed by IHC with an anti BrdU antibody, only cells that were in S phase during the labeling period will stain positively.
infect with a tetracycline inducible endothelial specific construct driving lacZ so you can pulse-chase the cells and just do a simple lacZ staining at the end point.
Hello everybody,
thanks for the nice answers and different ideas we can do this experiment. We now will think about which one will be the best for our conditions.
Thanks again!
Sven
Hi Santanu,
Does this dye ( tubulin tracker) irreversibly bind to tubulin and whether this is a reason for cell division arrest? if so are there chances that it may also affect migration, considering that cytoskeleton is also important for migration?
Hi Sven,
This is a good question, for which there are several potential solutions, both commercial and self-assembled.
My first response is to ask what instrumentation you have (or wish) to collect data? If you have time-lapse microscopy, this is simple but time-consuming solution. Don't forget to collect sufficient data to achieve a statistically representative analysis.
I have used CFSE, Cell Tracker Green and the PKH series dyes in time-lapse and end-point assays. They all work well, although each has pro's and con's, depending on how you run the assay. These are all fluorescence-based.
In terms of practicality (read simplicity and speed) I like the PKH series dyes; they label the cell membrane and have a high quantum yield – they are bright. However, as with all manipulations, the labelling procedure does affect cell behaviour. The 1:1 dilution between daughter cells is an advantage and allows direct determination of mitotic cycles.
CFSE is cheap and effective. It is cytoplasmic, and also dilutes 1:1 between daughter cells. However, fluorescein is subject to compensation issues (read autofluorescence interference).
Perhaps the method of choice is, however, GFP labelling. These are now available in many colours, so that you can track different cell populations within mixed cell samples. Are you able to transfect your cells with a GFP construct? They are available commercially.
Of course, all of these strategies require imaging instruments; microscope, scanner, CCD system, etc.
If you don't want the cells to divide, again there are several options available; however, then one must ask what is the biological relevance of your assay? Be aware that most cell cycle inhibitors disrupt the cytoskeleton, which is the motive force for cell migration!
You have some careful decisions to make!
Cheers
David L.
Would the fact that you cannot differentiate between mother and daughter cells actually be a problem? Unless your treatment affects cell division, cells should divide similarly in control and treated cells and therefore this would not interfere with your analysis - unless you are interested in analyzing individual cells. In this case live imaging, as suggested by someone else, would be the way to go, otherwise you can never be sure which cell has migrated how far.
Btw. preventing division with microtubule poisons is not advisable, since it will probably also interfere with migration of you cells.
There is also a possible way by monitoring the change in elasticity of the cell surface. New cells are typically softer than older cells. This maybe done by AFM in accoustic mode or optical tweezers.
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