analysis of yeast cell cycle - problem (Apr/25/2008 )

Hello!
Staining cells with PI
1.collect 1x107 cells, 3000rpm,5min remove the buffer, and resuspend them in 0.1ml of 1X RNase solution containing 1% Tritonx-100. Incubate the sample for 1 hour at 37°C with shaking
2. Centrifuge the cells, 3000rpm,5min and resuspend them in 60 ug/ml of freshly prepared pepsin solution. Incubate the sample for 5 minutes at room temperature.
3. Staining cells with PI(50 µg/ml PI ,2mg/ml Na citrate in 50 mM Tris-HCl )at 4°C in the dark for 30-120min.
i can not get good result,i want to know the reason
the result added here
[attachment=4595:szl.jpg]

Hi!
I'm not an expert, but there are several things that I don't understand. Your protocol is quite different than mine, but I guest that you take it from a reliable source.

1. I fix my cells and you doesn't.
2. I'm not sure if R2 window is well located. Do you try with the other cell population? You have more cells that you are not interested in?
3. You can also see PI in FL3, maybe is nicer...
4. You need to acquire more cells. In modfit, they recommend 20000-25000 cells.

I guest that it will help!

Hi sus12
Thank you for your advice.I read it just now because I have not came here for a long time.I fixed cells with 70% eathol and try many ways ,but it is a pity that I still have not got good result.I do not know why.Do you have the experence on analysis yeast cell cycle with FACS by PI stainning.Please send message to me if you have ,thank you.My e-mail:zhining1983@yahoo.com.cn