Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

Protocol for Freezing Mammalian Cells


  • Appropriate cold culture media (i.e. cRPMI for T cell lines, cIMDM for PBMC, cDMEM for attaching cells).

  • Sterile DMSO

  • Cells to be frozen.

  • Freezing vial (1.8 ml, Costar 2028)

  • Mr. Frosty controlled freezing container.


  1. Make up 20% DMSO/media mixture. Keep cold.

  2. Spin down cells at appropriate speed (T cell lines 5 minutes at 1200rpm, PBMC 7 minutes at 1700rpm etc.).

  3. Resuspend cell pellet in cold culture media, using half the volume needed. Keep on ice.

  4. Prepare freezing vials with name, date and cell number.

  5. Drop-by-drop add cold 20%DMSO/media to full volume.

  6. Aliquot into freezing vials and cap.

  7. Transfer vials into Mr. Frosty.

  8. Take Mr. Frosty to -70° C freezer.

  9. After overnight freeze, transfer vials to liquid N2 tank for long term storage.

  10. Make appropriate changes to liquid N2 storage log.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.