96 well microplate nuclear staining + DNA (PI) for FACS analysis

Perform all steps at +4°C, up to the fixation point.

Nuclear immunostaining.

1- Resuspend the cells in complete Phenol red-deficient medium at a convenient concentration (each microtiter well contains 300 µl and you may not stain less than 500.000 cells per well).
2- Place 0.5 to 3 millions cell per well of a U bottom 96 wells plate.
3- Centrifuge at  1000 rpm for 5 min
4- Discard the supernatant  in a basin 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 lysing buffer 1x (made from the 10x stock solution). Several companies sell this lysis buffer. Alternatively see J Immunol Methods 124: 103, 1989. Plain formalin or gluteraldehyde fixation makes the cells very sticky.
7- repeat steps 3 to 5
8- add 250 µl of PBS to each well
9- repeat steps 3 to 5  (centrifuge, discard the supn and vortex)
10- add 50µl of goat serum (1:10 in PBS) to each well and leave for 15-45 min. Alternative blocking sera may be used (e.g. human, providing that the same serum is added to the secondary antibody used). Blocking with milk should be avoided because antigenicity may be reduced.
11- fill up with PBS
12- repeat steps 3 to 5  (centrifuge, discard the supn and vortex)
13- add the diluted antibodies (50 to 100 µl/well) and incubate for 45 min to overnight. Do not exceed 24 hrs of incubation because the formalin bonds begin to become loose thereafter.  Anti BCL-6 antibody PGB6 may be used 1:10 from the supernatant. Do not use anti BCL-6 polyclonal antibodies because the results may be unpredictable.
14- repeat steps 3 to 5  (centrifuge, discard the supn and vortex)
15- add 250 µl of PBS to each well
16- repeat steps 14 and 15
17- add the secondary biotin-conjugated antibody (50 to 100 µl/well) and incubate for 45 min. Protect from direct light exposure. Do not exceed 24 hrs of incubation because the formalin bonds begin to become loose thereafter.
A FITC-conjugated antibody may be used at this step, instead of a biotin-conjugated one.
18- repeat steps 14 and 15.
19- repeat steps 3 to 5  (centrifuge, discard the supn and vortex)
20- add Avidin-FITC diluted in biotin-free medium.
 Avidin-FITC (Vector) 1:300.

AFTER THIS STEP, PERFORM ALL PROCEDURES AWAY FROM DIRECT LIGHT.

21- repeat steps 3 to 5  (centrifuge, discard the supn and vortex).
22- repeat steps 3 to 5 with PBS instead of medium
(centrifuge, discard the supn and vortex).
23- add 50-100µl PBS containing 1U/ml of RNAse (DNAse-free) (Boehringer,  cat n° 1399-861, stock of 200U/ml).
24- incubate at 37°C for 30 min.
25- chill on ice.
26- add Propidium Iodide at the final concentration of 50 µg/ml .
 (Boehringer,  cat n° 1399-861, stock of 500µg/ml). Note that the higher the PI concentration, the stickier the cells. However, after this type of fixation, PI needs more time or concentration to stain DNA than with ethanol-fixed cells.
27- incubate at RT for 30 min to 1 hr, in the dark.
28- transfer to FACS tubes (Falcon, n° 2008) and top up to 300µl with PBS.

resuspend each well and transfer to 300 to 500 µl of PBS in a FACS tube.