Ethanol fixation for flow cytometry

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

Fixation for flow cytometry with ethanol. This method is very simple, quick and allows samples to be stored for weeks at –20°C.

  • From a 35mm-10cm plate harvest cells with trypsin into a 15ml tube.

Keep media as this helps to inactivate trypsin and many mitotic and dead cells will be floating in the media

  • Pre-chill 100% -20°C high grade Ethanol
  • Centrifuge at low speed 1500rpm 10 minutes to pellet
  • Remove supernatant media and resuspend in 1ml PBS
  • Transfer to a 1.5ml eppendorf
  • Centrifuge at 5000rpm for 2-5min to pellet
  • IMPORTANT: Remove supernatant PBS and gently resuspend pellet in 300ul of PBS
  • Add 700ul of 100% -20°C high grade Ethanol (EtOH) inverting tube a few times to ensure proper mixing and a good fixing of cells.
  • Store at –20°C until required for flow cytometry/FACS

This method is based, with permission, on an original protocol available here.