BrdU for human cells

Using Boehringer Mahnein Kit cat. #1296 736, or Roche kit cat # 1 296 736. (Same kit)

Not provided with the kit: Cell culture media, 50mM glycine, IS blocking solution and DAPI.

1- Grow cells on appropriate substrate and under appropriate condition.

2- Aspirate cell culture medium and add BrdU labelling medium:

BrdU labelling medium: dilute BrdU labelling reagent (bottle 1 from the kit) 1:1000 in sterile cell culture medium (final concentration 10 mmol/l)

3- Incubate cells at 37C, for 12 hours (S1 cells) (the time will depend of the cells and individual requirements)

4- Aspirate BrdU labelling medium.

5- Wash 3 times (or more) with washing buffer (=1X PBS)

Washing buffer: dilute washing buffer concentrate (=10X PBS) (bottle 2 from the kit) 1:10 with redist. Water.

6- Fix the cells : a) or b)

a- With 70% ethanol (in 50mM glycine pH2.0) for at least 20 min. (1hr is suggested).

b- With cold Methanol:Acetone

7- Wash 3 times with washing buffer (=1X PBS)

8- Blocking : a) or b)

a- Virginia used the Pierce blocking solution

b- 10 % GS in 1X PBS for 1hr, if EHS the same + Fab #152 1:100 for another hour.

9- Wash 3 times with washing buffer (=1X PBS)

10- Cover the cells with anti-BrdU working solution, and incubate for 30 min. at 37C. Prepare the working solution shortly before use.

anti-BrdU working solution: Dilute anti-BrdU solution (bottle 4 from the kit) 1:10 with incubation buffer (bottle 3 from the kit)

11- Wash 3 times with washing buffer (=1X PBS)

12- Cover the cells with anti-mouse Ig-fluorescein working solution, and incubate for 30 min. at 37C. Prepare the working solution shortly before use.

anti-mouse Ig-fluorescein working solution: Dilute anti-mouse Ig-fluorescein stock solution (bottle 5 from the kit) 1:10 with incubation buffer (bottle 3 from the kit)

anti-mouse Ig-fluorescein stock solution : has to be reconstituted with 1 ml of redist. Water before the first utilisation (stable at 4C)

13- Wash 3 times with washing buffer (=1X PBS).

14- Cover the cells with DAPI (1:10000 in PBS) for 3-5 min.

15- Wash 3 times with washing buffer (=1X PBS).

16- Cover with an appropriate mounting medium.