Dear Jiyuan,

1. Depends on cell type and stimulation, some cells are AICD sensitive and some are resistance.

2. Anti CD28 is co-stimulation along with TCR and can activate survival signal.

3.Dead cells non specifically absorb antibodies so whatever positive staining you get with dead cells are false positive.

Hope this will help you,

Regards,

Gajendra

Thx a lot Gajendra, excellently helpful answers.

Do you think it is necessary to evaluate and report the percentage of dead cells besides gating them away from the scope? I mainly worry about if it is safe to assume that the dead cells are evenly distributed as cytokine-positive and -negative groups and simply gating away the dead cells won't compromise the percentage evaluation of cytokine-positive cells.

best,

jiyuan

I agree with Gajendra.

T cells undergo AICD when incubated with strong stimulants esp. your +ve controls (PMA+ionomycin, PHA, SEB etc.). Here the extent of cell death might not be a big deal since this control just shows that you cells are viable and functional.

I do understand your concern that AICD could mean most of your cytokine secreting cells are dead and are not even being looked at since you just gated them out. However, I don't think it is necessary or useful to evaluate and report the % of dead cells. If you would like to minimize AICD, you could test different doses and different incubation times and choose a dose and an incubation time that result in little cell death but still gives you a good enough cytokine signal in Flow.

In my assays, I use PMA+ionomycin and see around 80% of cells dead by the time I terminate the cultures. I still see 50-60% of CD4+ T cells being +ve for IFN-g in the live gate while my test stimulants induce very little cell death (20%) but the frequencies of CD4+ IFN-g+ cells in the live gate (80%) are

Thanks julie, splendidly useful answer, and now I completely understand gadendra‘s view.

I guess I can end this question now.

again appreciate the help from you two.