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

  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 successive 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.
tracking

 

 

 

 

 

 

Sample histogram using this protocol.

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