The extensive tyrosine dephosphorylation is an intrinsic part of apoptotic process of haematopoietic cells and may be involved in the apoptotis induced by different stimuli. It seems that activation of one or more phosphatases is an intrinsic part of the suicide program in haematopoietic cells.
 

GENERAL PROCEDURE

Brdu Staining

Cells are cultured in the presence of 10-50 m M 5-Br-2dU and (Sigma Cat# B-5002) At least 4 hours is necessary for optimal signals.

The cells can be surface stained with directly conjugated mAb before fix and perm.

Cell fixation and permeabilization

Add 200 m l of PBS with 2% paraformaldehyde, 1mM EDTA, and 0,1% saponim, 2mM NaVO₄ (Sodium orthovanadate) (Sigma) to the cell pellet. Mix well and incubate at room temperature for 10 min.

Wash once in PBS 1% FCS/or 1% BSA, wash twice if using microwell plates.
Remove supernatant carefully leaving 20 m l.

Stain cell

The cells can be stained with Phosphotyrosine (Py 20 direct PE conjugate) and incubate on ice for 30 min and wash once afterwards with PBS.
Add 300 m l PBS with 0.5% paraformaldehyde.
Leave at RT for at least 4h to stabilize the binding of mAb to cell surface and cytoplasmic antigens.

DNA-Brdu stain

Add 22 m l PBS with 5% Tween 20 per 100 m l PFA solution.
(Tween 20 permeabilizes the nuclear membrane)
Leave overnight at 4C.
Wash twice in PBSC leaving 20 m l of fluid when removing supernatant.
Make a solution of 20 mg/ml DNAse I (Sigma Cat. DN-25) in PBS (room temperature).
Per Sample: 10 m l DNAse solution 3 m l anti-BrdU FITC (Becton Dickinson)
Leave for at least 1 h @ room temp.
Add 10 m l PBS with the DNA dye (either 7 aminoactinomycin D Molecular Probes Cat. no. A-1310 or 100 m g/ml. (AAD stock solution: 1 mg 100 m l MeOH then into 900 m l PBS).
Leave for 15 min. Ten add 200 m l of PBS.