2- Place 0.25 to 3 millions cell per well of a 96 U bottom wells plate.
3- Centrifuge at 1500 rpm for 5 min (below the 4.5 tag on the big bench centrifuge)
4- Discard the supernatant in the sink with a quick wrist-forearm movement.
5- Vortex the whole plate in order to resuspend the cells in the few µl left.
6- Fix for 10 min in 200µl Becton Dickinson lysing buffer 1x (made from the 10x stock solution).
7- repeat steps 3 to 5 (centrifuge, discard & vortex)
8- add 250 µl of PBS to each well
9- repeat steps 3 to 5 (centrifuge, discard & vortex)
10- add 100µl of goat serum to each well and leave for 15-45 min.
11- fill up with PBS
12- repeat steps 3 to 5 (centrifuge, discard & vortex)
13- add the diluted antibodies (50 to 100 µl/well) and incubate for 45 min to overnight
14- repeat steps 3 to 5 (centrifuge, discard & vortex)
15- add 250 µl of PBS to each well
16- repeat steps 14 and 15 (centrifuge, discard, vortex, fill-up)
17- add the secondary antibody (50 to 100 µl/well) and incubate for 45 min. Protect from direct light exposure.
18- repeat steps 14 and 15 (centrifuge, discard, vortex, fill-up).
19- repeat steps 3 to 5
20- add 50 µl of 10% buffered formalin to each well, protect from light and store at +4°C
21- resuspend each well and transfer to 300 to 500 µl of PBS in a FACS tube.
;)