Hi,
I wanted to analyze cell cycle for HaCaT by flow cytometry with propidium iodide staining. After fixation, do the cells incubate at 37 degree for 30 minutes initially with RNAase A followed by PI at room temperature for 30 minutes or do the cells in PI/RNAase mixture solution and incubate at 37 degree for 30 minutes?
TIA
Hello Sudheer Babu,
Incubate the cells in PI/RNAase mixture solution and incubate at room temperature for 30 minutes.
You prepare a 10ml of 0.1% Triton X-100 solution in PBS and to it you add 2mg RNase A and 200ul of 1mg/ml PI.
1. Fix the cells in 70% ethanol for at least 1 hour at 4 degree C.
2. Centrifuge the ethanol fixed cells 5 min at 300 x g and decant ethanol thoroughly.
3. Suspend the cell pellet in 5 ml PBS, wait 60 sec, and centrifuge 5 min at 200 x g.
4. Suspend the cell pellet in 1ml PI staining solution. Keep at either 37°C for 15 min or at 30 min at room temperature.
Best,
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