> Can anyone advise on lysis buffers which (i) will and > (ii) will not permeabilise (for, say, PI > exclusion/access), and similarly on fixation which would > or would not permeabilise - and any combinations of > lysis/fixation to have these opposite effects. With regard to fixation: If you fix with 0.5% paraformaldehyde, then PI exclusion is maintained for about 2 hours. We typically stain our cells with about 1 ug/ml PI before fixation, wash the cells, and then fix. You have to analyze on the FACS within a couple of hours; by 6 hours, the signal begins to degrade and the "negatives" begin to show significant PI staining. By 24 hours, it's basically worthless. If you want to do live/dead on cells that you will permeabilize, use ethidium monoazide bromide (EMA) at 5 ug/ml, wash, and expose to light for 10 min. It's nowhere near as good as PI, and because live cells pick up significant stains, you must devote a channel (usually, FL3) to this discriminant. (Unlike PI, which is compatible with immunofluorescence staining on the same channel). mr
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