Hello everyone. Recently, I have been struggling with T cell proliferation assay. I found that the CFSE signal (peak) of T cells did not decrease after anti-CD3/28 stimulation. However, I can still see some spheroids in the culture plate. In my view, the spheroid formation is a way to know the cell proliferation. Thus, I want to know what is the reason for this difference and how can I fix it?

And my protocol is

1. Prepare a 96-well flat bottom plate coated with anti-CD3 in PBS under RT for 3 hours.

2. Seed 2*10^5 CFSE-stained CD8+ T cells in RPMI-1640 complete media with further soluble anti-CD28 (the ratio of CD3:CD28 is 5:1) and beta-ME addition .

3. Culture for 5 days.

4. Detect the CFSE signal (peak) by flow cytometry.

Note: CD8+ T cell will be isolated from PBMCs, which keep on ice for 2-3 hrs due to the experiment of neutrophil treatment.

Another following question is I also culture T cells with neutrophils for the immunosuppression assay. However, my result showed that the CFSE+ ratio is increased after neutrophil co-culture compared with T cells only (these two groups have CD3/28 stimulation). Is it possible?

Thanks for replying !

Sincerely, Zhen jie