Mullins Molecular Retrovirology Lab

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

Protocol for Thawing Mammalian Cells


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

  • 50 ml conical tubes (thaw max 3 vials /tube).

  • Sterile plastic and glass Pasteur pipettes.

  • 10 and 25 ml pipettes.

  • Vial(s) with cells to be thawed.


  1. Perform procedure swiftly, minimizing the time cells are in warm 10% DMSO media.

  2. Thaw cells by putting vial in 37° waterbath, until just a small ice-clump remains.

  3. Using sterile Pasteur pipette, transfer the cells from vial into labeled 50 ml conical.

  4. Slowly add the cold media and shake tube gently while adding in the media. Dilute 10% DMSO media at least 1 in 20.

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

  6. Aspirate media carefully, using sterile glass Pasteur pipette.

  7. Resuspend cell pellet in 5 ml of appropriate media.

  8. Use sample for counting.

  9. Make appropriate changes to liquid N2 storage log.