Posted 24 May 2011 - 08:21 AM
my protocl is using mixstaining solution(1uM YO-PRO+10ug/mlPI+Hoechest(33342(1:500) ) to incubate myoblasts for 30min in 37degree incubator, and then wash with PBS, and then use 4% PFA to fix for 15min in 37degree incubator. and then wash with PBS 2x. Finally,i checked on the fluorecent microcope. The Hoechst33342 is fine, the nuclei are very clear ans sharp. But when i shift to GFP channel, several cells are green, but not clear, their nuclei are disfussion, and clear and sharp. The red ones are the same as the green. And there are very strong background. So i can not distiguish which one is apoptosis.
So i would like to know what's wrong with my protocol? Also, many cells are detached because i finish staininig and then fix.I think I already did evry gentlly. And how to get rid of background?
I am looking forward to your suggestions. I am in hurry for waiting...
Posted 24 May 2011 - 06:38 PM
Posted 25 May 2011 - 02:44 AM
Try washing more after the fixing, you may be seeing free formaldeyhde autofluorescence.
Thank you for reply. I will try 3 times washing. Hopefully, it will help get rid of background.
Posted 25 May 2011 - 05:37 PM
Posted 26 May 2011 - 03:51 AM
If you are still stuck, try washing in 0.1 M glycine in PBS - it apparently mops up free aldehydes. Autofluorescence can be a problem too, there are ways around it with peroxide and other things, but we can deal with that later if needed.
Thank you. And i will try.
But i have 2 questiones here.
1.http://www.cbm.uam.es/confocal/Manuales/Ioduro%20propidio.pdf. From this link,why need do RNAase treatment??? I know Propidium iodide are from different company.
2. How do you think my mentioned protocol? It is ok or not? I just take a reference for flow cytometry protocol.
Posted 26 May 2011 - 05:18 PM
It should be fine for FACS, which uses lasers to determine the fluorescence output, this is much more specific for the dyes than using a mercury burner such as those found on microscopes. You probably won't even need to do any more washes before FACSing