Cell cycle analysis using propidium iodide and BrdU


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Reagents

Protocol

  1. Treat 1 x 106 cells with 10 uM BrdU for an appropriate time (10 minutes - 48 hours, depends on the cells. 30-60 mins seems a good starting point.).
  2. Harvest and wash (1500 rpm, 5 mins)
  3. Fix with ethanol: NB: attendant clumping problems. Several methods here vary from rapidly adding 70% ICE-COLD ethanol to adding 100% ethanol at -20 degrees C dropwise while vortexing to get a final concentration of 75%. Volume should be around 4 ml. Critical thing is to KEEP THINGS ICE COLD. Do the fixation in polypropylene tubes e.g. 15 ml screw-cap. Don't fix with paraformaldehyde, this spoils the CVs.
  4. Store in ethanol at 4 degrees for at least 4 hours or up to several weeks.
  5. Spin off alcohol and wash x2 with PBS; faster spin nornally needed (2000 rpm, 5 minutes). Transfer to Eppendorf tubes on last wash. DO NOT USE POLYSTYRENE TUBES as the cells will stick to the sides.
  6. Resuspend cells in 2M HCl (1 part conc HCl added to 4 parts DI water). Leave at room temperature for 30 mins with periodic agitation/vortexing. this step denatures the DNA. Better results may be obtained with 4M HCl.
  7. Spin off acid and wash x2 with PBS-T (PBS + 0.5% BSA + 0.2% Tween 20). Alternatively, wash x1 with 0.1M sodium tetraborate, pH 8.5. Important to remove all the acid because it interferes with the antibody staining in the next step.
  8. Add 20 ul 1/10 dilution anti-BrdU antibody (Becton-Dickinson) directly to cell pellet and incubate 20 mins at room temp in the dark. ** For negative control - omit the antibody **
  9. Wash x2 with PBS-T
  10. Stain with 50 ul 1/50 dilution of FITC-conjugated rabbit anti-mouse (F(ab')2 fragments preferred: DAKO) for 20 mins at room temp in the dark.
  11. Wash x1 with PBS.
  12. Resuspend cells in 1 ml cocktail containing PI at 20 ug/ml and RNAse at 100 ug/ml (Sigma). Incubate 30 mins in the dark at room temp or overnight in the fridge. NB: Recommended PI concentrations vary from 2.5 to 50 ug/ml. Polystyrene tubes (Falcon 2052) are okay at this point.
  13. Store at 4 degrees until analysis.

References

http://science.cancerresearchuk.org/sci/facs/facs_major_apps/cell_cycle_analysis/bromodeoxyuridine_staining/facs_brduprotocol/?version=1 http://www.pathology.washington.edu/research/labs/rabinovitch/flowroom/protocols/RabLabManual.html#_Toc94006943 http://imaging.altervista.org/flowcytometry_protocols/brdu_cellcycle.html http://www.people.cornell.edu/pages/jls269/protocols/cellcycleprotocols.html http://www.bdbiosciences.com/immune_function/pdfs/02-8100055-21A1_Ch9.pdf http://sciencepark.mdanderson.org/flow/files/BrdUPI.pdf http://www.fhcrc.org/labs/fero/Protocols/BrDU_PI.html