1. Wash cells in PBS and resuspend 2 x 106 cells in 0.5 ml PBS.
2. Fix cells by addition of 5 ml of 1% paraformaldehyde. Incubate on ice for 15 min.
3. Wash cells twice and resuspend in 0.5 ml PBS in polypropylene tubes.
4. Permeabilize cells by addition of 3 ml of ice-cold ethanol (70%) for at least 4 hr on ice.
(Cells may be stored for several weeks in 70% ethanol at -20oC)
5. Centrifuge sample, wash in PBS and resuspend cells in 50 µl of TdT reaction solution:
10 µl TdT reaction buffer
2.0 µl BrdUTP stock solution
15 U TdT, 5µl CoCL2 (10 mM)
33.5 µl H20
6. Incubate cells for 40 min at 37oC with occasional mixing (or room temp. overnight).
7. Add 1.5 ml rinsing buffer and centrifuge.
8. Add anti-BrdU-FITC antibody (approx. 100 µl) and incubate at room temp. for 1 hr.
9. Add 1ml of PI staining solution, incubate for at least 30 min at room temp. in the dark.
10. Analyse for FITC/PI fluorescence with appropriate controls for instrument setup.
The exact incubation times for fixation, permeabilization, TdT may vary according to cell type. It may be necessary to optimize these for specific cells and applications.
TdT Reaction Buffer (5X):
1M potassium or sodium cacodylate, 125mM TRIS-HCl, pH 6.6, 1.25mg/ml BSA. Store at -20oC.
Anti-BrdU-FITC Antibody Solution:
0.3 µg anti-BrdU-FITC antibody, 0.3% w/v Triton X-100, 1% BSA, PBS to 100 µl volume per test.
PI Staining Solution:
Propidium Iodide 5 µg/m PI, RNase A (DNase free) 200 µg/ml, PBS, pH 7.2-7.4.
Many vendors sell kits with pre-prepared buffers and reagents. Alternatively, individual reagents may be purchased separately. The above protocol is a general guide and individuaql kits may vary in the amounts of reagents used.