A simple method for the isolation of platelet-free lymphocyte suspensions from rat whole blood

A simple technique for obtaining platelet-free lymphocyte suspensions from rat whole blood for use in complement-dependent microcytotoxicity assays is presented. Rat lymphocytes were purified by filtering buffy coat preparations containing thrombin over nylon fiber columns prior to Ficoll-Hypaque centrifugation. The addition of thrombin to buffy coat preparations significantly (P < 0.001) enhanced platelet retention on the nylon fiber columns. This procedure yielded lymphocyte suspensions which had a 98% mean reduction in platelet contamination compared to lymphocyte preparations which were purified using only Ficoll-Hypaque centrifugation. The thrombin-nylon fiber filtration method produced lymphocyte suspensions containing an average of 98% lymphocytes, and the total yield of lymphocytes isolated from 2 ml of blood averaged 2.85 X 10(6) cells. The sensitivity of complement-dependent microcytotoxicity assays was increased by the use of the thrombin-nylon fiber-filtered platelet-free lymphocyte preparations compared to the use of platelet-contaminated lymphocyte suspensions.