FACS experiment in tubes

Author: Orsolya Symmons

Date: 2015-04-29

Comment: this protocol seems to gnerate more sample-to-sample variability than the plate setup.


  • Make sure you have enough FACS buffer (PBS+10%FCS+0.01g NaN3) and PFA.
  • Count cells. Take 4 x106 cells per cell line
  • spin cells @4ºC, 400g 5min
  • meanwhile prepare MM of antibodies (650µl FACS buffer+6.5 x antibody (based on FACS calculation sheet))
  • remove medium. Resuspend each sample in 400µl ice-cold FACS buffer.
  • Split each tube into 4x 100µl.
  • Add 100µl FACS buffer (unstained control) or 100µl antibody master-mix
  • Put in cold-room 30min, rotating
  • spin cells @ 400g, 4ºC 5min
  • resuspend cells in 500ml FACS buffer
  • spin cells @ 400g, 4ºC, 3 min
  • resuspend cells in 200µl ice-cold PBS
  • to each tube add 200µl 4% PFA
  • keep in cold room 20'
  • after fixation, spin cells @ 400g, 4ºC, 5min
  • wash with 500µl ice-cold PBS
  • spin cells @ 400g, 4ºC, 3min
  • resuspend cells in 750µl DAPI solution (PBS+10%FCS+0.1% Triton-X 100+1µg/ml DAPI)