...
- 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 cells to remove clumps (pipette up and down)
- Transfer suspended cells to 15 mL Falcon Tubes
- Centrifuge cells 5min 2500 rpm
- Resuspend cells in ice cold PBS, 10% FBS, 1% sodium azide to a concentration of 5X10^6 cells per mL with a total volume of 2000 uL
NOTE: DO NOT ASPIRATE SOLUTIONS CONTAINING SODIUM AZIDE. Pipette waste into an empty bottle and dispose of it appropriately later. - Add primary antibody in optimal concentration (Dilutions should be made in PBS
- Incubate on ice for 30 minutes to 1 hr
- Centrifuge cells 5min 2500 rpm
- Resuspend cells in 2000 uL PBS 10% FBS, 1% sodium azide
- Centrifuge cells 5min 2500 rpm
- Repeat 10 & 11 two more times
- Add the secondary antibody at optimal concentration (MINIMIZE LIGHT EXPOSURE)
- Incubate IN THE DARK! for 30 minutes to 1 hr
- Centrifuge cells 5min 2500 rpm
- Resuspend cells in 2000 uL of ice cold PBS, 3% BSA, 1% Sodium Azide
- Repeat 14 &15 two more times
- Transfer suspension to cytometry tubes
- Run flow cytometry