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  • Vial of frozen cells
  • 37°C Water Bath
  • Hank’s Balanced Salt Solution (HBSS)
  • Centrifuge
  • Tubes, Pipets (all solutions must be endotoxin-free)

Neutrophils require a different approach to cell thawing and handling because they are so easily activated and damaged. A few key steps will ensure high viability.


  1. Place vial of cells in 37°C water bath and agitate until thawed. It is important to thaw the cells quickly. Do NOT allow thawed cells to remain in freezing media any longer than necessary.
  2. In a 15 mL conical tube, slowly add freshly thawed cells to 9 mL of HBSS. DO NOT USE MEDIUM-CONTAINING SERUM. Invert tube 2 or 3 times to mix, or mix gently by pipetting up and down several times.
  3. Centrifuge for 10 minutes at 200 x g.
  4. Aspirate or decant the supernatant and gently resuspend the cell pellet in 10 mL of HBSS.
  5. Remove an aliquot for cell count and proceed with experimental manipulations.
  6. Neutrophils can be activated by endotoxin, excessive agitation, or repeated centrifugation with cell death resulting. Use of polypropylene tubes is recommended. Minimize centrifugation steps and use promptly after thawing.