White Blood Cell Cryopreservation Protocol May 2005

- February 2006 -

Blood is collected in purple top tubes which contain EDTA as the anti-coagulant. The tubes are centrifuged at 800 x g for 20 min so that the sample is separated into plasma (top layer), buffy coat (middle layer; white blood cells), and erythrocytes (bottom layer; red blood cells). An aliquot of plasma (800μl) is placed in a tube with 200μl of dimethyl sulfoxide and the remainder of the plasma is set aside for use in the next step. The buffy coat is then removed, measured, and diluted to 1 ml with the extra plasma. The buffy coat sample is then diluted with the dimethyl sulfoxide/plasma sample. The remainder of the plasma is then disposed of in accordance with the Hazardous Materials Disposal Protocol of the laboratory. Care should be taken in these first steps to minimize the amount of red blood cells present in the samples. If completely clear (no red blood cells) samples is desired, then after the buffy coat has been removed it, and the plasma can be diluted in phosphate buffered saline, in separate tubes centrifuged at 800 x g for 10 min, and the buffy coat and clear plasma supernatant can be removed. The plasma is then added to the dimethyl sulfoxide and the buffy coat is diluted as described previously.

The samples can then be loaded into 0.5 mL and maintained at 5ºC until freezing. Cryopreservation of the white blood cells is performed using a programmable freezer with the following freeze curve: 5ºC to -40ºC at -1ºC/minute, -40ºC to -85ºC at -4ºC, and then plunge the samples into liquid nitrogen for storage. Samples are thawed for 30 sec in a 37ºC water bath and immediately placed on ice until used or processed further.

References

Truax, R.E. et al., 1993. Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies. Am J Vet Res 54:862-866.

Kleinschuster, S.J. et al., 1979. Cryopreservation of bovine monomuclear leukocytes. Am J Vet Res 1649-1651.