Titrating Antibodies (Mouse)  

  • Use appropriate cell suspension for marker being titrated
  • Following cell count, add 3x10^6 cells/test to a 96 well plate
  • Centrifuge at 1300rpm for 3mins
  • Remove supernatant
  • Add 30uL of antibody mixture. Antibodies are made up in FACS buffer

Normal Titrations are:

  • 1/50
  • 1/100
  • 1/200
  • 1/500
  • 1/1000
  • If very concentrated may need to be titrated further
  • Allow to stain for 15-20mins in the fridge (Preferably in the minifridge to avoid contact with light)
  • Add 150uL FACS buffer to dilute stain, then centrifuge at 1300rpm for 3mins
  • Remove supernatant in plates by flicking upside down over a sink (check cells have pelleted before so you dont loose your cells!)
  • For biotinylated antibodies, now add an appropriate secondary conjugate and repeat. For the direct conjugates (only 1 step staining) resuspend in 200uL FACS buffer and transfer into FACS tubes for acquisition

Note: Unless a secondary antibody is required, no compensation tubes are needed. A Cells alone tube is however, always required.