Hello everyone,
I'm trying to use PI staining to determine cell cycle with flow cytometer. I use PI/RNase Staining Buffer (BD, 550825) and follow their protocal (http://www.bdbiosciences.com/documents/BD_FlowCytometry_DNA_Staining_Protocol.pdf).
However, my ethanol fixed cells all lyse immediately after I add PBS. I called their technical support but they are not helpful at all. Does anyone know how to solve this problem?
Thanks!
Hi Fang,
This may be a silly question, but are you 100% certain you are not accidentally using 10X PBS instead of 1X PBS? It is a very easy mistake to have happen.
Nicole
Hi Nicole,
Not silly at all! But I'm 100% sure I used 1X PBS. Actually I've tried PBS from different sources. I've also tried to use DPBS, HBSS. They all give me the same result.
Fang
Hi Fang, your issue is kind of puzzling. Your ethanol-fixed cells are already lysed. Ethanol dissolves your cells lipids, thus disrupting their membranes. It would not be even possible to get an osmotic effect afterwards.
If you want to perform an incubation in pbs, I imagine you could rehydrate them in a series of alcohol dilutions of decreasing concentration. Best of luck,
D
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