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.