Fernandez L A, MacSween J M, Robson D A
Department of Medicine, Camp Hill Hospital, Halifax, Nova Scotia, Canada.
Immunol Lett. 1989 Aug;22(2):167-71. doi: 10.1016/0165-2478(89)90185-5.
One of the methods of obtaining B cell colonies involves physically admixing B cells in growth medium containing mitogens with accessory T cells treated with mitomycin C or low dose irradiation to prevent T cell colony formation. However, under these conditions T cells still have the capacity to form colonies in methylcellulose, which is most frequently used for colony assays, and B cells recovered from the colonies are contaminated by the large number of added T cells. Therefore, we have developed a method of growing B cell colonies by physically separating the enriched B cells from the T cells by using millicell-HA inserts (Millipore). The requirement for mitogens, culture conditions, development, and number of B cell colonies formed were similar to those observed with admixture of T and B cells. The advantage of this system is that it permits growth of B cell colonies without the confounding influence of T cell proliferation or treatment of T cells with mitomycin or irradiation, and relatively pure B cell colonies can be recovered for further characterization or functional studies.
获得B细胞集落的方法之一是将含有丝裂原的生长培养基中的B细胞与用丝裂霉素C处理或低剂量照射的辅助性T细胞进行物理混合，以防止T细胞集落形成。然而，在这些条件下，T细胞仍有能力在最常用于集落测定的甲基纤维素中形成集落，并且从集落中回收的B细胞会被大量添加的T细胞污染。因此，我们开发了一种通过使用密理博-HA插入物（密理博公司）将富集的B细胞与T细胞进行物理分离来培养B细胞集落的方法。对丝裂原的需求、培养条件、B细胞集落的发育和形成数量与T细胞和B细胞混合时观察到的情况相似。该系统的优点是它允许B细胞集落生长，而不受T细胞增殖或用丝裂霉素或照射处理T细胞的混杂影响，并且可以回收相对纯的B细胞集落用于进一步的表征或功能研究。
