Thawing Neutrophils

Materials

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

Procedure

  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 pipeting up and down several times.
  3. Centrifuge for 10 min 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.

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.