


Protocol for immunophenotyping with direct
and indirectly conjugated mcabs
- Aliquot equal
numbers of cells from a cell pellet or 100 ml
of whole blood into Falcon tubes.
- Add the required indirectly
conjugated mcab (1 mg) to each aliquot of
cells.
- Mix on Vortex.
- Incubate at RT for
20 min.
- Wash cells in PBS
if using isolated cells.
- If using whole
blood add 1 ml of 1:10 diluted Becton Dickinson FACS Lysing Solution.
- Mix on Vortex.
- Incubate at RT for 10 min.
- Aspirate off supernatant.
- Add 0.5 mg of secondary conjugated antibody to pellet of
cells.
- Mix on Vortex.
- Incubate at RT for
20 min.
- Wash cells in PBS.
- Aspirate off supernatant.
- Add the directly
conjugated mcabs (1 mg) to each aliquot of
cells.
- Mix on Vortex.
- Incubate at RT for
20 min.
- Wash cells in PBS.
- Resuspend in <
0.5 ml PBS.
- Collect least
10,000 events.