Hewitt C W, Sawyer S, Beck P A, Martin D C
J Immunol Methods. 1980;36(3-4):227-34. doi: 10.1016/0022-1759(80)90128-3.
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.
本文介绍了一种从大鼠全血中获取无血小板淋巴细胞悬液用于补体依赖的微量细胞毒性试验的简单技术。在进行Ficoll-Hypaque离心之前，通过在尼龙纤维柱上过滤含有凝血酶的血沉棕黄层制剂来纯化大鼠淋巴细胞。向血沉棕黄层制剂中添加凝血酶可显著（P < 0.001）增强血小板在尼龙纤维柱上的保留。与仅使用Ficoll-Hypaque离心纯化的淋巴细胞制剂相比，该方法产生的淋巴细胞悬液中血小板污染平均减少了98%。凝血酶-尼龙纤维过滤法产生的淋巴细胞悬液平均含有98%的淋巴细胞，从2 ml血液中分离出的淋巴细胞总产量平均为2.85×10⁶个细胞。与使用含有血小板污染的淋巴细胞悬液相比，使用凝血酶-尼龙纤维过滤的无血小板淋巴细胞制剂可提高补体依赖的微量细胞毒性试验的灵敏度。
