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)