Hello everyone,
I am trying to improve my CFSE assay of culturing mouse CD4 T cells. I separated mouse whole CD4 T cells with beads, and culture in 96-wells. I used CD3/CD28 to stimulated the cells dyed with 0.5uM CFSE. After 4 days, I collected the cells and dyed with PI before the FLOW cytometry detection. But the results were horrible and not normal. I tried many times and can not figure out the reason. Can someone help me about this puzzle?
Thanks.
A bit late to the party, but I think your CFSE concentration is your main problem. I agree with Dmitry, the amount of counted cells is not very high. And indeed, like you said yourself, the compensation should not be the problem. Like Jinbo Yu mentioned, it is due to the staining.
To monitor the proliferation of human T-cells, I used a CFSE concentration of 10 microM and measured CFSE dilution after three and six days. I received beautiful peaks at both time-points. Before that, I used 1 microM and received very bad peaks at both time-points, hence the changeover. During my experiments, non stained cells appeared around log2-3 in the histogram. Taking that in consideration, it appears like your CFSE histograms are showing a false positive result as did my initial experiments with 1 microM CFSE.
So increasing the concentration of CFSE is the most important change you have to make in your assay. There has to be something to dilute. I used 10 microM but less could suffice.
Finally, you should also use a low flow-rate during your measurements. I don't know your current, but it should be low. This will create more distinguishable peaks in your histogram.
Good Luck,
IJs
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