The following protocol combines BrdU (FITC) staining of S phase cells in combination with propidium iodide for cell cycle analysis.

This protocol is used courtesy of Prabhat Goswami, Radiation Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa.

Stock Solutions

  • All solutions should be prepared in distilled and autoclaved water.
  • Pepsin (Sigma - P-6887-1g) in HCL:[100 ml of 2N HCL and 20 mg pepsin].  Make pepsin fresh
  • 0.1M Borax, Na2B4O7, (pH 8.5).
  • PBT (PBS+1mg/ml BSA+0.1% Tween20) Always fresh

BrdU Pulse-Chase Conditions

Start with at least 2 x 106 cells per milliliter. Alcohol fixation and multiple wash steps reduce the final number of cells. The final target concentration for analyzing with flow cytometry is 1 x 106 cells per ml.

  1. Add 100 microliter of BrdU (1mM stock; Sigma-5-bromo-2-deoxyuridine, B-5002-1 g) per 10ml media and incubate for 30 min at 37oC.
  2. This step is only when doing pulse chase:
    Wash the monolayer with sterile warm PBS (two times) and continue culture in regular growth medium supplemented with cytidine and thymidine.
  3. At representative time, trypsinize and centrifuge cells (1200 rpm in J6 centrifuge for 5 min at 4oC).
  4. Wash cell pellet with cold PBS, spin, and add 1 ml cold 70% ethanol and store at 4oC. 
    Samples good for up to a month.

Sample preparation for flow cytometry

  1. Centrifuge samples (1200 rpm in J6 centrifuge for 5 min at 4oC).
  2. Resuspend in PBS.
  3. Centrifuge as before.
  4. Resuspend pellet in 3 ml PBT. 
  5. Centrifuge as before.
  6. Repeat above wash step.
  7. Resuspend pellet in 1 ml 0.2 mg/ml pepsin prepared in 2N HCl and incubate at room temperature for 30 min.
  8. Neutralize mixture by adding 3 ml 0.1 M Borax.
  9. Centrifuge (1500 rpm for 5 min at 4oC).
  10. Wash pellet with 3 ml PBT.
  11. Centrifuge (1500 rpm for 5 min at 4oC).
  12. Repeat wash.
  13. Pour off supernatant and resuspend pellet by finger vortexing.
  14. Add 55 microliter BrdU primary antibody (5 microliter antibody +50 microliter PBT) and incubate at room temperature in the dark for 60 minutes. Shake the tube occasionally.
  15. Add 2ml PBT and centrifuge (1500 rpm for 5 min).
  16. Wash pellet with 2ml PBT and spin.
  17. Pour off supernatant and resuspend pellet by finger vortexing.
  18. Add 55 microliter FITC-conjugated secondary antibody (5 microliter FITC-conjugated anti-IgG in 50 microliter PBT) and incubate for 60 min at room temperature in the dark. Shake the tube occasionally.
  19. Wash and centrifuge (1500 rpm for 5 min) as in step 16 (twice).
  20. Add 100 microliter freshly boiled and cooled RNAse A (1 mg/ml stock prepared in PBS; Calbiochem-Ribronuclease A #55674 50 TU), mix gently and incubate 30 min at room temperature in the dark.
  21. Add 0.5-1.0 ml propidium iodide or a volume appropriate to for a final concentration of 1 x 106 cells per ml (35 microgram/ml stock prepared in PBS) and incubate for 60 min at room temperature in the dark.
  22. Filter samples using 70um nylon mesh.
  23. Analyze on flow cytometer with 488nm excitation and bandpass emission filters for FITC (530/30) and propidium iodide (585/42) or (610/20). Samples can be stored overnight at 4oC for next day analysis.

References

Sarsour EH, Agarwal M, Pandita TK, Oberley LW, Goswami PC. Manganese superoxide dismutase protects the proliferative capacity of confluent normal human fibroblasts. J Biol Chem. 2005;280(18):18033-41. PMID: 15743756.