Cell Cycle analysis using propidium iodide and BrdU
Reagents
- PBS
- PBS-T: PBS + 0.5% BSA + 0.2% Tween 20
- Ethanol
- HCl
- BrdU
- anti-BrdU antibody (Becton-Dickinson)
- FITC-conjugated rabbit anti-mouse (F(ab')2 fragments preferred: DAKO)
Protocol
- 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.).
- Harvest and wash (1500 rpm, 5 mins)
- 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.
- Store in ethanol at 4 degrees for at least 4 hours or up to several weeks.
- 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.
- 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.
- 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.
- 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 **
- Wash x2 with PBS-T
- 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.
- Wash x1 with PBS.
- 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.
- Store at 4 degrees until analysis.
References
