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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
    • 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.