The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Assessment of Cell Cycle

The following protocol is for Hoechst 33258 / Ethidium Bromide staining of cultured cells for assessment of cell cycle.

Reagents

  • 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.

Staining

Staining of cultured cells for assessment of cell cycle

Sample histogram using this protocol.

  1. At the start of the culture, add BrdU (5'-bromodeoxyuridine) (0.3mM) and 2' deoxycytidine (1 x 10-4M) to cell cultures.
  2. At the time of harvest, centrifuge cells in 1ml eppendorf tubes and resuspend in 1ml of RPMI +10% FCS + 10% DMSO.
  3. 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.
  4. Thaw cells. Centrifuge cells at 4°C and aspirate supernatant.
  5. 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.
  6. Add 1.5µg of ethidium bromide for 15 minutes at 4°C.
  7. Run on flow cytometer.

See chapter 21 of Methods in Cell Biology, volume 41 for more detail.