Proliferation assay

[spillou]

Hi everyone,

I am trying to assess my cell proliferation by using a CFSE assay, however I can't obtain more than one single pic on the flow cytometer after 5 days, even if I know that my cells did proliferate.
Does anyone have an idea about it?

Thanks a lot

[biohazard]

The most obvious reason is that there's something wrong in the CFSE staining you've done. Double-check your protocol that you did all correctly. If I recall correctly, CFSE is very sensitive to light, so you must protect it or else some few hours exposure can spoil it.

Btw, what methods you use to ensure that your cells have prolified? Do you just check them under a microscope or count them in a chamber, or just look at the colour of your medium?

What cells do you use? For example T cells need proper stimulants to make them divide (e.g. PHA if you want unspecific proliferation), as well as the feeder cells. It is easy to think your cells proliferate, when the actual metabolism and medium colour change is caused by the feeder cells, not the cells you want to study. (And you cannot count the T cells directly because of the feeders)

You could provide me some more information about your cells and the protocol, and I can try to come up with alternative solutions.

[spillou]

Thanks a lot for your reply.

I am using primary T-cells indeed, but I tried with some MM6 macrophages and K562 (to check if the cell type might make a difference). I am stimulating my T-cells with PMA instead of PHA, but as these twomitogens are pretty similar, I don't think this make a huge difference.
I am counting my cells on a Neubauer chamber with trypan blue, and I am also using a nucleocounter to get the number of cells/their viability.

I wasn't aware of the CFSE light sensitivity. I will protect it from the light from now.

About the CFSE concentrations, I diluted it quite a lot (down to 500 times the lowest concentrations proposed by the manufacturer) and also used it highly concentrated, but it doesn't make any difference (well apart from the GMFI of my single pic obviously).

If you have any ideas, please let me know, because I really have no other clue.

Thank you.

P.S: I bought two kits as I was supposed to use it quite a lot, and I have the same results with both batches

[biohazard]

Aight, let me know if protecting from light fixed the problem - that's what caused some CFSE staining problems in our lab. If it's not exposure to light that ruins your experiment, then I'll try to figure out what else it might be.