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Cell cycle analysis for SubG1 fraction


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#1 speci

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Posted 18 September 2013 - 07:01 AM

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

speciBildschirmfoto 2013-09-18 um 16.59.12.png

 

Here I induced cell death:

Bildschirmfoto 2013-09-18 um 16.59.18.png

 



#2 bob1

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Posted 19 September 2013 - 03:42 PM

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



#3 theo22

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Posted 30 September 2013 - 04:19 AM

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



#4 speci

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Posted 04 October 2013 - 09:04 AM

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!






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