Freezing Cells for FACS

Reagents:

  • 70% DMEM/10% DMSO/20% FCS  or
  • 90% DMEM/10% DMSO/1mg/mL BSA

Note: FCS will improve cell viability, but should not be used when cell sorting as it may alter gene expression and cell bioactivity. Maintaining cell viability is an issue with this protocol, and the user should test the freezing process before proceeding.

 

Protocol:

  • Prepare a single cell suspension as per normal.
  • Resuspend cells at ~1-2x107 per mL in freezing media.
  • Aliquot cells into 2.0 mL cryo tubes. Place tubes into freezer boxes for slow freeze. Store at -80°C until ready to stain.
  • When needed, thaw cells quickly using a 37ºC water bath and then immediately transfer to ice.
  • Transfer cells to 15 mL conical tubes and wash once in the buffer to be used for FC/FACS (i.e. PBS/2 mM EDTA with either 2% FCS or 1 mg/mL BSA).
  • Proceed to block Fc receptors and stain as normal.

Note: Ensure buffer is ice cold and that all future steps occur at 4°C/on ice.