Hello all,

I have done PI cell cycle analysis for one of my cell line of interest. Briefly, I have harvested the cells with trypsin EDTA, and added ice-cold alcohol (70%) on to the pellet and incubated on ice for 30 minutes. Further, two washes were given followed by addition of 100ul PBS and 10ul RNase to the pellets and incubation @ 37 in a water-bath for 30 minutes followed by addition extra PBS and FACS acquisition. 10ul PI from stock (1mg/ml) was added just before the acquisition. After the acquisition I have analyzed the data. Below are the gating that I have taken step wise

1. Gating on SSC-A vs FSC-A (P1) to get rid of debris

2. Gating on PE-A vs PE-W (P2) to get rid of the doublets

But I found extra clusters above the P2 and gated it as P3 . I am not sure what it represents , is that a diploid continuum or aneuploidy?

I have attached the figures herewith. Hope some one can guide me on this.

Regards

Aravindan Narayanan