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Propidium Iodide Staining for FACS Analysis
- Add 400 µl (300 µl) culture to 1.2 ml (0.9 ml) 100%
ethanol in Eppendorf (volume not critical, therefore depends how
much culture can be spared).
- 1 h-2+ weeks at 4° C or –20° C.
- Spin down cells.
- Prepare n x 1 ml SSC (n = number of samples).
- From that, prepare n x 0.25 ml SSC + 0.25 µl RNaseA.
- Resuspend cells in 250 µl SSC RNase by vortexing.
- Incubate at 50° C overnight (or at least 1 h if in a rush
— but overnight gives better results).
- Prepare n x 50 µl SSC + 1.5 µl proteinase K.
- Add 50 µl SSC protK to samples.
- Incubate for exactly 1 h at 50° C.
- Prepare n x 0.5 ml SSC + propidium iodide (final concencentration
is 3 µg/ml).
- Add 0.5 ml SSC propidium iodide to samples.
- Incubate at room tempearture for 1+ h then store at 4° C
until use. (Overnight or over weekend gives best results but can
store for at least a month and still get results.)
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