...
- 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 warm cold complete media.
- Triturate cells to remove clumps (pipette up and down) to disperse clumps.
- Pipette cell suspension into eppendorf tubes
- Centrifuge for five minutes
- 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).
- Transfer suspended cells to Cytometry tubes
- Centrifuge cells
- Resuspend cells in ice cold PBS, 10% FBS, 1% sodium azide to a concentration of 1-5X10^6 cells per mL with a total volume of 2000 uL
- Add primary antibody in optimal concentration at optimal time (listed on order form) (Dilutions should be made in 3%PBS)
- Centrifuge cells
- Resuspend cells in 2000 uL PBS
- Centrifuge cells
- Repeat 10 & 11 two more times
- Add the secondary antibody at optimal concentration for an optimal time (INCUBATE IN THE DARK!)
- Centrifuge cells
- Resuspend cells in 2000 uL of ice cold PBS, 3% BSA, 1% Sodium Azide
- Repeat 14 &15 two more times
- Run flow cytometry Run FACS.