Immunostaining for Flow Cytometry

STEPS:

Note: Number the wells and the corresponding tubes ( the FACS machine deals with numbered samples better). Record the contents of the wells to which the numbers correspond

1. Aspirate off media.
2. Rinse wells with 500uL of PBS/versene. Aspirate.
3. Add 200uL of trypsin/EDTA to each well. Incubate at room temperature for 90 seconds, then neutralize with 800uL of warm complete media.
4. Triturate (pipette up and down) to disperse clumps.
5. Pipette cell suspension into eppendorf tubes 
6. Centrifuge  for five minutes
7.  
8. 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).
9. Run FACS.