You don't need to wash the cells many times, the less the better, PBS should be ice cold and you need to keep the cells on ice all the time except centrifuging. You can also try to shorten the PI staining period.

I agree with Xin Ren and can also suggest using less PI staining reagent i.e. 50% try to avoid vortexing the tubes are much as possible. If you see the strong differences in your data, but they are affected by the high PI background of your untreated cells you can try to subtract the PI-positive cell values of the untreated cells from the experimental group, but of course, another experiment is preferable.

hi

I agree with xin & Anton

1- you should add the 1or 2 ul PI to 100ul test tube ,3 min before the assay by FACS

2- check the viability your cells before the test ( mycoplasma free cell line )

3- use positive( Staurosporine or ...treated)and negative condition(with out treated) for your test

good luck