I want to do cell cycle mess with facs. Is it better for permeabilization to use 70% methanol or 70% ethanol? How long should I incubate with meth/eth at 4C? What concentration Pi should I use and what incubation time?
Dear Matthias,
You can do either but both ethanol and methanol must be ice cold when fixing and kept on ice for 1 hour.I used a final concentration of 10ug/ml from a 1mg/ml stock of PI.
Hope this helps.
Incubated in 4 degrees in dark for 10-20 mins.You can standardize whats best for you.Dont forget RNase A.
wash cells wit pbs. fix cells with 70% chilled ethanol on voretx. and keep in -20 for at least 2 h then add 400uL of solution ( 25ug/ml of RNase+ 40ug/ml of PI+ 0.03% of NP40 ) mix vigorously and incubate at 37 0C for 30 min. Acquire directly.
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