Optimization ProcedureThe following steps may be necessary to determine the suitable concentration of each reagent:
1. Prepare dead cells by 10 minutes incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30 minutes incubation in 70% ethanol.2. Stain dead cells with 0.1-10 μM PI solution to find a PI concentration that stains the nucleus only, not the cytosol.3. Stain dead cells with 0.1-10 μM Calcein-AM solution to find a Calcein-AM concentration that does not stain the cytosol. Then stain viable cells with that Calcein-AM solution to check whether the viable cell can be stained.
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