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.
- Vial of frozen cells
- 37°C Water Bath
- Hank’s Balanced Salt Solution (HBSS)
- Tubes, Pipets (all solutions must be endotoxin-free)
- 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.
- 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.
- Centrifuge for 10 minutes at 200 x g.
- Aspirate or decant the supernatant and gently resuspend the cell pellet in 10 mL of HBSS.
- 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.