• Created by Unknown User (clrichar@mit.edu), last modified by Unknown User (lylaatta@mit.edu) on Aug 01, 2014 13:11
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 cold complete media
  4. Triturate cells to remove clumps (pipette up and down)
  5. Transfer suspended cells to 15 mL Falcon Tubes.
  6. Centrifuge cells 5min 500 xg at 4°C.
  7. 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. 
  8. Add primary antibody in optimal concentration (Dilutions should be made in PBS + 3% BSA)
  9. Incubate on ice for 30 minutes to 1 hr
  10. Centrifuge cells 5min 500 xg at 4°C
  11. Resuspend cells in 2000 uL ice-cold PBS
  12. Centrifuge cells 5min 500 xg 4°C
  13. Repeat 10 & 11 two more times.  The last time, resuspend in 500 µl ice-cold PBS + 10% FBS + 1% NaN3
  14. Add the secondary antibody at optimal concentration (MINIMIZE LIGHT EXPOSURE)
  15. Incubate IN THE DARK! on ice for 30 minutes to 1 hr
  16. Centrifuge cells 5min 500 xg 4°C
  17. Resuspend cells in 2000 uL of ice cold PBS.
  18. Repeat 14 &15 two more times.  The last time, resuspend in 500 µl ice-cold PBS + 3% BSA + 1% NaN3
  19. Count cells on a hemacytometer (not all of samples, just a few.)  Dilute to ~1e6 cells/ml with ice-cold PBS + 3% BSA + 1% NaN3. Transfer suspension to cytometry tubes.
  20. Run flow cytometry.
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