BrdU and PI staining for live cells and animals

(Ethanol - PFA method)

Safety warning: BrdU is mutagenic. Dispose carefully. Use gloves.

Positive controls:

grow a lymphoblastoid cell line in two aliquots, one supplemented with 10µM BrdU, 24 hr. Mix equal amounts of cells at 1:1 ratio. This should give a 50% positive peak for BrdU.

Cell lines:

pulse the cell lines with with 10µM BrdU, 1 hr. Wash thoroughly and keep on ice.

Live animals:

inject IP BrdU at 50 mg/kg in PBS, 1-2 hrs before killing. Note that fetal labelling may be suboptimal uder these conditions.
 Make a single cell/nuclear suspension from the organs to be analyzed.
 
1- wash with ice-cold saline
2- resuspend in  0.5 ml ice cold saline.
3- add 1.2 ml cold (-20°C) 95° ethanol
4- add 2 ml cold (+4°C) PBS.
5- centrifuge
6- resuspend in 1 ml 1% PFA
7- fix for 30 min RT
8- centrifuge and pour off supernatant
[8bis- take an aliquot of the cells to be stained without DNAse as negative control]
9- resuspend in 1ml DNAse 50 U/ml in PBS
10- incubate for 10 min. RT
11- wash in PBS
12- add blocking serum
13- add the diluted antibodies (50 to 100 µl/well) and incubate for 45 min to overnight. Anti BrdU (Becton Dickinson) works 1:200.
14- 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 antibody (50 to 100 µl/well) and incubate for 45 min. Protect from direct light exposure. BrdU staining and PI DNA staining only allow Goat anti-mouse FITC  counterstaining.
18- repeat steps 14 and 15.
19- centrifuge, discard the supn and vortex
20- add 50 µl of PI solution (0.1 mg/ml in PBS) and incubate for 30 min.
21- resuspend each well and transfer to 300 to 500 µl of PBS in a FACS tube.