1. Take cells directly from a culture flask into 50-mL conical tubes and centrifuge at 400g for 5 min.
2. Discard the supernatant and resuspend in medium (cell culture medium or phosphate-buffered saline [PBS] with 1% bovine serum albumin).
3. Centrifuge again at 400g and discard supernatant. Count cells and resuspend at an appropriate concentration, which will vary with sorter used but will be in the range of 1 X 106-1 X 107 per mL. The final suspension medium will depend on the cell types to be sorted. In general, a low protein concentration is recommended because this will lead to less cell clumping although the addition of 5 mM EDTA (ethylene-diaminetetraacetic acid) will also help this.
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