Hi,
The cell cycle experiment I am doing requires to distinguish dead cells from the HeLa cell population. For this purpose, I am using Zombie Green (ZG) viability dye (from BioLegend) before the fixation step. Then I stain the DNA content with Propidium Iodide and analyze with Flow. I am having a bit wary feeling about the ZG vs FSC-A plot I am getting as it does not seem to discriminate two distinct live vs dead cell population. Being a newbie to flow, I am having hard time to exclude dead cells out from my cell cycle experiment. I will highly appreciate your thoughts and suggestion on this. Thank you. (Attached is the plot)
You'd better talk to people in the flow cytometry facility of your institution. They must be experts, and they are there to help you.
Hey Chandra. Lots of questions like this are common when you first start out in flow. Like all good experiments proper controls in flow experiments are a must. Mixing a heat killed sample with live cells and then staining with ZG would show you exactly where dead/live cells are within your plot. Other thoughts: What do you expect your viability to be? Cell count can help with this, before and after treatment. Sometimes FSC vs SSC will provide some general info on cell condition. DNA histograms can offer some clues about dead cells as well. Use everything to your advantage when determining a positive population, but most import is to include gating controls. Hope that helps!
Remember that PI stains both DNA and RNA.
Thank you Matthew and Shin-Ichiro for your inputs. I highly appreciate it.
Would be interested to see how you went with this. I am using ZG in platelets without DNA therefore cannot counter PI stain for conformation. Would be interested if you could provide a workup? Cheers Evan
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