Labelling of Nuclei with BrdU and PI for FACS

This protocol uses acid tratment to denature the DNA. Other protocols use thermal denaturation which is more efficient and therefore more sensitive for BrdU incorporation. However, thermal denaturation results in greater cell loss. The following protocol works well for Rat 1a cells.

  1. Fix cells in 70% EtOH as for standard PI staining.
  2. Centrifuge at 3.5K in microfuge for 10min at 4¼C. Discard supernatant.
  3. Resuspend pellet and add 200ul of 2M HCl/Triton -100 (this will cause aggregation of the cells). Incubate at room temperature for 30mins to denature the DNA. Mix gently every 10min-Tap a little.
  4. Centrifuge at 3.5K in microfuge for 10min at room temperature.
  5. Aspirate off supernatant, resuspend pellet and add 200ul of 0.1M Na2B4 O7 , pH 8.5 to neutralize the acid.
  6. Count the cells and transfer ~1x105 to an eppendorf tube. If the pellets look similar, just count 1 or 2.
  7. Centrifuge cells at 12,000G for 10sec and aspirate off supernatant.
  8. Resuspend cells in 20ul of PBS + 0.5% Tween 20 + 1% BSA and add 10ul of anti-BrdU FITC (Becton Dickinson, Cat. No. 7583) per 1x106 cells. Incubate for 30min at room temperature in the dark.
  9. Spin down cells at 3.5K in microcentrifuge for 10min, aspirate off supernatant and wash in 50ul of PBS +0.5% Tween 20 + 1% BSA.
  10. Resuspend cells in 200ul 38mM NaCitrate + 69uM propidium iodide. Add 1ul of 9.5mg/ml RNaseA (19ug/ml final). Incubate for 30min at 37¼C in the dark.
DFCI FACS 632-3179