BrDU Staining Protocol

BrdU Staining Protocol

Supplies

·       Ethanol 100% USP (highest quality)

·       FACS Staining Buffer (1XPBS w/ 3% calf serum, 0.05% sodium azide-filtered) —Dilute staining antibodies in Buffer

·       DNase (Sigma D-5025, Bovine Pancreas)

·       RNase (Boehringer, 25 mg bovine pancreas)

·       Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow)

·       0.15 M NaCl, 1.5 M NaCl

·       10% Paraformaldehyde (kept as stock in -80°C)

·       Tween 20

·       1M MgCl2

·       FACS Tubes

Protocol

Cells in 96-well FACS plate

1.    Block with 24G-2

2.    Surface stain cells as usual

o   Omit fourth channel labeled antibodies on all stains; EtOH destroys APC

3.    Prepare tubes from which to transfer EtOH drop wise (1.2 ml EtOH on ICE)

4.    Resuspend cells from 96-well plate with 100 µl 0.15M NaCl (cold)

5.    Transfer to FACS tubes ON ICE. Add 400 µl 0.15M NaCl to each tube

6.    Vortex at 1/3 speed and add EtOH with pasteur pipette at 1 drop per second. This is a critical step… do not add EtOH too quickly

7.    Incubate on ice for 30 minutes

8.    Spin 10 minutes @ 2000 RPM, 4°C

9.    Dump and shake liquid into waste

10.Using repeat pipetter, add 1 ml FACS staining buffer into each tube

11.Spin 10 minutes and dump as before (step 8)

12.Add 1 ml 1% paraformaldehyde + 0.05% Tween 10

For 20 ml:

2.0 ml 10% paraformaldehyde 10 µl Tween 20

13.Incubate at room temperature for 30 minutes

14.Incubate on ice for 30 minutes

15.Spin and dump as before (step 8)

Add 1 ml DNase (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units/ml DNAse)

For 50 ml:
46.5 mL dH20
200 ul MgCl2 (1M stock)
1500 uL NaCl (5M stock)
100 Kunitz units DNAse (volume depends on activity of batch)
Incubate for 30 minutes @ 25°

16.Spin 10 min. and dump as before (step 8)

17.Transfer cells from FACS tubes to 96-well plate. Wash once with staining medium.

18.Block with 10% rat serum. Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical to spin at high speed once the cells have been fixed with EtoH/PFA since they become less dense).

19.Add anti-BrdU-FITC or biotin (1:20 dilution for Phoenix flow).

20.Pipette up and down to resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4°C).

21.Wash and dump as before. Transfer cells into FACS tubes.