apoptosis assay - immunofluoresence staining (May/24/2011 )

I am going to use Propidium iodide(sigma4170), Hoescht and YO-PRO-1 (Y3603)dyes to stain myoblast cells for apoptosis assay. Myoblast is growing in to 6-well collagen I coating plates.

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...

My eamail:sfyang3511@gmail.com

Try washing more after the fixing, you may be seeing free formaldeyhde autofluorescence.

bob1 on Wed May 25 02:38:37 2011 said:

Thank you for reply. I will try 3 times washing. Hopefully, it will help get rid of background.

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.

bob1 on Thu May 26 01:37:15 2011 said:

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.

PI, like other nucleotide intercalating dyes will also intercalate with RNA, usually at a much lower amount per length of RNA than would be found on DNA as the RNA is single stranded mostly, whereas DNA is double stranded and the dyes intercalate into the morphology of the dsDNA better than they do the ssRNA. Basically what I am saying is that there is some signal from the RNA when you use PI. As you want the signal to be from the DNA, use a RNAse to get rid of the RNA.

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