Cavaillon J M, Cinader B, Chou C T, Dubiski S
J Immunol Methods. 1979;26(1):1-10. doi: 10.1016/0022-1759(79)90036-x.
B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.
通过去除与木瓜蛋白酶处理的兔红细胞形成玫瑰花结的细胞,从兔脾脏中分离出B淋巴细胞,使其不含T细胞。如果在用去除玫瑰花结的方法进行分级分离之前,先用磁铁去除粘附或摄取聚L-赖氨酸包被的铁芯颗粒的细胞,可实现进一步的纯化。通过分别用针对RABELA和RTLA的抗体对B细胞和T细胞进行补体介导的细胞毒性杀伤来评估细胞产量和纯化效果。其他标准取决于测定携带Fc受体的细胞数量,以及用刀豆球蛋白A、PHA或针对受体Ig轻链同种异型特异性的抗体刺激的细胞的胸腺嘧啶摄取量。以约占原始脾脏中B细胞20%的产量获得了纯化的B细胞制剂,其中非B细胞含量不到10%。该方法可实现不干扰分离细胞细胞膜的纯化。