1- Resuspend BM, spleen and FL cells in azide-free, Phenol-red-
and biotin-free medium supplemented with 10% heat-inactivated FCS. Keep
on ice.
2- Lyse Red Blood Cells with 1 ml RBC lysing medium for
2 min at RT.
3- Add 5-7 ml of cold medium.
4- Layer a cushion of .5 ml of FCS at the bottom of the
tubes and centrifuge.
5- Resuspend the cells in medium. (practically, top up
to the desired volume with medium)
6- Aliquot the cells (1-10 X106) in 0.5 ml
aliquots in Eppendorf tubes and warm at 37°C for 15 min.
7- Prepare the 2x hypotonic shock medium: FDG 2mM, Verapamil
100µM, in water. Warm at 37°C.
8- Mix equal volume of cells in medium with the 2x hypotonic
solution.
9- Place for 2 min at 37°C.
10- Add 1 ml cold PBS-serum-azide (PBS-S). Chill. Spin
down and resuspend in cold PBS-S.
From now on keep on ice constantly and use only PBS-S.
11- Place 0.25 to 3 millions cell per well of a 96 U
bottom wells plate.
12- Centrifuge at 1500 rpm for 3 min (below the
4.5 tag on the big bench centrifuge)
13- Discard the supernatant in the sink with a
quick wrist-forearm movement.
14- Gently vortex the whole plate in order to resuspend
the cells in the few µl left.
15- Proceed with the immunostaining.
RBC lysing medium:
Ammonium chloride (NH4Cl) 8.29 g 0.155
M
Potassium bicarbonate (KHCO3) 1 g 10mM
EDTA 0.037 g 1mM
H2O 1l.
Filter with 0.45µm filter
aliquot in 100 ml aliquots
filter the solution to be used with 0.2µm filter.