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- MATERIALS: Ensure that trypsin is completely thawed, but PBS/versene and complete media are cold (do NOT place in warm water bath).
- Aspirate off media.
- Rinse wells with 500uL of PBS/versene. Aspirate.
- Add 200uL of trypsin/EDTA to each well. Incubate at room temperature for 90 seconds, then neutralize with 800uL of cold complete media.
- Triturate (pipette up and down) to disperse clumps. Transfer to cytometry tubes. Place tubes in ice (cells must be kept cold; in stasis).
- Count the cells from 2-3 tubes, using the hemacytometer (typically 2x10^6 per mL). Add PBS to dilute the cells to 1x10^6 per mL (typically, an additional 1mL is required).
- Run FACS.