Hi, which antibody/marker acts best to distinguish between MEFs and mESCs in flow cytometry when using Annexin V-PI for Apoptotic and also for PI only cell cycle analysis?
Considering that Annexin V is a surface marker, whereas for PI only- in cell cycle- the cells are fixed with 70% ethanol overnight.
Many thanks,
Pooja.
Hi Pooja,
I use to analyse mESCs on FACS with PI and SSEA1. If you need it I can send you the full protocol for this purpose.
Cheers
Pierre
Hi Pierre,
Thanks a lot for your reply. If you can share your protocol, that would be great! Just to get it clear, you used SSEA1 as a stem cell marker, along with PI for cell cycle?- I've been having some troubles in getting distinct G0 and G1/G2 peaks in cell cycle.- I think I'm messing up something in fixing the cells. Any suggestions/ recommendations?
Thanks again.
Pooja.
Hi Pooja,
No worries. Yes it is a protocol to stain with SSEA1 and PI together. Use the SSEA1-AF647 from SantaCruz as you don't want to overlap the PI. Here it is:
1) Harvest 1 million mESC, 2) Stain w/ SSEA1 - AF647 (SC-21702 - 1/100th). 3) Fix 1h w/ 1%PFA at 4C, 4) PBS wash, 3) Add 1mL EtOH 70% -20C vortexing 5) let cells at least 2h00 at -20C. 6) Wash twice 7) Add RNAse A (0.1mg/ml) 30min at 37C. 8) Add PI (0.02mg/mL) 9) FACS.
For the FACS, I don't know your skills so sorry if I say basic things you may already know:
-Assess the cells at 200 events / sec max as you want to distinguish 2n versus 4n. It will be very slow to process cells but you need that to be accurate.
- It is not exponential scale as GFP, so change scale to "linear" .
-You can also put less PI and adjust the voltage.
-Also don't adjust the voltage on the FACS and leave. During the analysis more and more PI integrate DNA so the fluorescence may increase, and you may have to adjust the voltage between samples.
I hope it helps
Cheers
Pierre
Great! Thanks a ton Pierre! I'll try it out and let you know how it goes..
Cheers!
Pooja.