We are doing cell cycle analysis in ZR-751 cell lines. We seed cells @ 5x105 cells per well in six well plate on day 1. On day-3 we are treating cells with DNA damaging agent for 1 hour followed by either no time for recovery or 2 hour, 8 hours and 24 hours of recovery time in fresh media. After trypsinisation and washing with 1x PBS I used following protocol All centrifugations were carried @ 210g for 5 minutes @ 4C.
1-Fixation in 3.7% formaldehyde PBS 10 minutes @ RT.
2- 3X PBS washing
3- Permeabilisation with 0.1% triton 100X, 3 minutes @ RT, washing 3X with PBS.
4- We added 20 ul of 10mg/ml RNASe and 200 ul of 50ug/ml propidium iodide per sample.
5- After 45 minutes in dark at Ice, cell cycle analysis was carried on FACS canto.
We are having really weird results attached file.
Thanks for suggestion!
on your dot plot, there are some debris and dead cells should be excluded, then on your FSC-A vs FSC-H plot, the tail will disappear; and you can add PE-A vs PE-H plot, gating out the tails which means duplets; after these two steps, your cell cycle plot should be more readable.
I used to do cell cycle analysis after irradiation of my cells. As mentionned Daniela, I always used ice cold EtOH 70 for fixation and then storage at -20.
Every time after irradiation I obtained this kind of "weird results" (done with facsDIVA too). I find it very interesting that you observed the same thing with a DNA damaging agent (which should be same effect but chemically).
The only idea I got is that maybe your treatment (as for irradiation) induces an alteration of DNA compaction (and not of the content) due to the gamma-P-H2AX signaling. And this DNA compaction modification alters the staining of IP not uniformly (stoichiometry reaction).
Thanks everyone!
@ Sylvain Ferrandon, Then how do you result analysis because it is quite hard to adjust gating for sub-population?
I am using Bleomycine that causes DNA induction.
You can eventually try a co-staining with BrdU-FITC to determine the cells in S-phase. By using two different labelling, you will be able to discriminate more accurately.
Good luck!
Hello Abdul,
In addition to the above answers, I will suggest you to fix the cells in 90% ehtanol overnight at -20 after trypsinisation and one wash with PBS and then adding RNAse PI next day, giving an incubation for 15 minutes and acquiring the cells in FACS. You can use RNAse PI solution from BioProbe and it works very well. You will get separate peaks as per your expectation.
Hope it helps !
Disclaimer: I am not intended to support any company product and this is my personal experience
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