Protocol for immunophenotyping with direct and indirectly conjugated mcabs

 

  1. Aliquot equal numbers of cells from a cell pellet or 100 ml of whole blood into Falcon tubes.
  2. Add the required indirectly conjugated mcab (1 mg) to each aliquot of cells.
  3. Mix on Vortex.
  4. Incubate at RT for 20 min.
  5. Wash cells in PBS if using isolated cells.
  6. If using whole blood add 1 ml of 1:10 diluted Becton Dickinson FACS Lysing Solution.
  7. Mix on Vortex.
  8. Incubate at RT for 10 min.
  9. Aspirate off supernatant.
  10. Add 0.5 mg of secondary conjugated antibody to pellet of cells.
  11. Mix on Vortex.
  12. Incubate at RT for 20 min.
  13. Wash cells in PBS.
  14. Aspirate off supernatant.
  15. Add the directly conjugated mcabs (1 mg) to each aliquot of cells.
  16. Mix on Vortex.
  17. Incubate at RT for 20 min.
  18. Wash cells in PBS.
  19. Resuspend in < 0.5 ml PBS.
  20. Collect least 10,000 events.