Cell cycle analysis for SubG1 fraction - (Sep/18/2013 )

Hey all,

I'm currently doing cell cycle analysis to reveal dead cells via the SubG1 fraction (one aspect of several other apoptosis assays I do).I do the following PI staining protocol:

- Induce cell death

- harvest cells (adherent cells)

- resuspend pellet in staining solution (0.1% Triton X-100, 50ug/ml PI, 0.5x PBS).

- keep on ice until flow cytometry

I get a real nice cell cycle if I don't induce cell death. I observe a strange cell population in the samples where the cells start do die.

This population is someting between the S-Phase and G2-phase population (see image). In the FSC/SSC plot they appear as a very low SSC and medium FSC population. Have you ever seen this?

Best Wishes

This is the control = No cell death induction

speci

Here I induced cell death:

I suspect it will be debris with some DNA incorporated in it.

These are your apoptotic cells.

Apoptotic cells will bind less PI and shift to the left. So G1 cells will appear as a subG1 peak. But G2/M cells that go into apoptosis also shift to the left and they appear as if they are S-phase cells. However, they are just aopototic G2/M cells.

When you give apoptotic cells a color in a PI-/PI-W dotplot, you will see where these cells appear in your FSC/SSC plot. I did this many times and observed that apoptotic cells have the behaviour you also see in the FSC/SSC plot

Thanks for your answer!

I also suspect that these are my apoptotic cells. But the following is quite strange: If i induce apoptotis with a different agent (Calcium Ionophor), I only have a Sub-G1 population and not a SubG2 like above. Is it possible that my first protocol induces apoptosis in all cell stages, including G1 and G2 and therefore I get this two subpeaks? On the other hand, the Calcium Ionophor induces apoptosis only in the G1 stage, therefore I get only SubG1?

This sound logic to me, but I'm worried because I can't find a publication where they also have these two Sub-Peaks. It's hard to claim this without any reference. Do you have some references as you've seen this already @theo22?

Thanks again!