The University of Iowa Carver College of Medicine
Assessment of Cell Cycle
The following protocol is for Hoechst 33258 / Ethidium Bromide staining of cultured cells for assessment of cell cycle.
- Hoechst Staining Buffer (prepare in water)
- 0.1M Tris (pH 7.4)
- 0.154M NaCl
- 0.5mM MgCl2
- 1mM CaCl2
- 0.1% NP-40
- 0.2% BSA
Add Hoechst 33258 (2µg/ml) to the staining buffer just before the buffer is added to the sample.
Sample histogram using this protocol.
- At the start of the culture, add BrdU (5'-bromodeoxyuridine) (0.3mM) and 2' deoxycytidine (1 x 10-4M) to cell cultures.
- At the time of harvest, centrifuge cells in 1ml eppendorf tubes and resuspend in 1ml of RPMI +10% FCS + 10% DMSO.
- Freeze sample at -20 °C for 1 hour minimum. Cells may remain frozen until samples from sucessive days can be run on flow cytometer at one time.
- Thaw cells. Centrifuge cells at 4°C and aspirate supernatant.
- Resuspend in 1ml of ice cold Hoechst staining buffer and incubate at 4°C for 15 minutes in the dark. Cell density should be between 4 x 105 and 1 x 106 cells per ml.
- Add 1.5µg of ethidium bromide for 15 minutes at 4°C.
- Run on flow cytometer.
See chapter 21 of Methods in Cell Biology, volume 41 for more detail.