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
- 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 Falcon 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
- Transfer suspension to cytometry tubes
- Run flow cytometry