Could you please tell me if I use the correct gating strategy for Brdu analysis, where the agent X makes my cells fail to incorporate Brdu after one hour of incubation, when compared to the control untreated cells. Yet, the DNA content, based on simultaneous PI staining, shows that many cells are in S phase. The calculated percentages of the cells in each cell cycle phase by gating G0/G1 and G2/M Brdu-negative and S phase Brdu-positive cells obviously contradicts the predictions made by the FlowJo software, based only on PI staining curve (see attachment). If I would have used only PI staining, I may have made a wrong conclusion, that my cells are still proliferating, when they are not, since they do not incorporate Brdu, right?
Do my gates G0/G1 and G2/M need to touch each other? Or should I include the fourth gate to indicate the cells that fall in S phase by DNA content, but do not incorporate Brdu? Does that suggest that the cells have been arrested in S phase when I began the treatment? I am quite a novice in flow cytometry so any feedback would be appreciated. Many people use the manual calculation strategy, but please take notice of the fact that the data are different from that calculated by FlowJo and in one case are less reliable than in the other. This example makes me wonder, how can I trust non-Brdu data in other experiments?
- Badges
- Science method