The use of cryopreserved lymphocytes for measuring cell-mediated immunity to allo-antigens in dogs by a microcytotoxicity assay

Lymphocyte — mediated cytotoxicity of frozen stored cells against target allogeneic fibroblasts was compared to that of freshly prepared lymphocytes in a microcytotoxicity assay. Peripheral blood lymphocytes obtained from alloimmune dogs were suspended in cold pooled dog serum containing 10% dimethyl sulfoxide, slowly cooled in a GV Planer R 201 programmed freezer and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were rapidly thawed, dimethyl sulfoxide was diluted out of the sample, and the cells were suspended in culture medium containing 20% fetal calf serum. Cytotoxic activity of cryopreserved lymphocytes was only slightly affected by the freezing procedure.