Hi,
Guys, I performed cell cycle analysis using PI staining recently. The problem is the compound I am working should lead to cell cycle arrest and the data suggests that. But, this is not enough. (Data is attached; D24 and D48 compound treated samples for 24 and 48 hours).
What I see is that in compound treated cells and non-treated cells, the majority of cells are G1 phase. I can indeed see increase in cells in G1-phase in compound treated cells (suggesting G1/S arrest). But, the cells in G1 phase originally is too high already (nearly 70%).
Can someone suggest me error in my protocol?
1. Seed cells (24 hours)
2. Serum starve cells (24 hours)
3. Add compound treatment and DMSO (negative control) 24 or 48 hours)
4. Wash cells with PBS 2X
5. Treat cells with Trypsin-EDTA and collect cells and centrifuge at 300g for 5 minutes.
6. Remove media and add 4-5 mL (PBS+2%FBS )
7. Fix cells by adding 3-4 mL 70% ethanol. Make sure to vortex mildly while adding ethanol. Carefully check if they are no clumps being formed. Make sure to use pipette for continuous aspiration. Incubate for 1-2 hours at 4C on ice. Cells could be kept at -20C for weeks.
8. Add more PBS+2%FBS and centrifuge at 500g for 5 mins.
9. Wash 1 more time in PBS+2%FBS and centrifuge for 5 mins at 500g.
10. Add solution with 0.5 ug/ml RNAase in PBS (with 0.1% sodium citrate and 0.1% Triton-X 100) for 30 mins at 37C. Total solution volume is 0.5 mL
11. Add PI staining solution (100 ug/mL) 0.5 mL solution and keep at 4C overnight. It is suggested to vortex gently in between a couple of times.
Regards,
Pratik
Read about how to use double thymidine block to get better results.
Hi Pratik,
Unfortunately I cannot see your images.
My PI staining protocol is a bit different from yours, because I usually stain for 1 hr prior to process cells, but I believe anyway that is not the main issue.
Since you reported a potential G1 arrest also in your control cells, I would check the cell density to avoid overgrow. In addition, you should keep in mind that starvation likely leads to cell cycle arrest. You did not specify which compound you are employing, so I can't tell if starvation is necessary for this treatment. I would anyway try to set up again the experiment with in parallel the same samples without starvation. If that is the cause of G1 arrest in control cells, you should be able to see that.
Good Luck
Hi,
I already did samples without starvation. The results were even more worse than with starved samples. So, I was confused.
You can use softwares like FLOWjo or MODFIT to analyze my data.
Regards,
Pratik
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