I am planning to do flowcytometry on isolated cells from specific brain regions. Since I get a few thousand cells from 4-5 mice, I am thinking of fixing (formalin-methanol and store in -20degreeC) and pooling them over a period of time. Will this have an effect on sorting and more importantly, I am planning to do RNA analysis from the sorted population of cells.
Fixing:
- 10% formalin (4% formaldehyde in PBS-DEPC) 10min room temperature followed by 1 min on ice incubation
- Adding methanol to the cells so that the final concentration of methanol is 90%
- On ice for 30 min and stored in -20degreeC
Can I save the cells in 90%methanol at -20C for like a month so that i can get enough cells for sorting? Does that affect the RNA in anyway?
Anton Lennikov
It is pretty much depends on the proteins you are interested in studying. It might work.
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Aloysius Wilfred Raj Arokiaraj
Thanks Anton. I am planning to study the transcriptome.
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