Morley A A, Trainor K J, Seshadri R S
Exp Hematol. 1983 May;11(5):418-24.
A simple technique for cloning lymphocytes at limiting dilution in microwells has been developed using phytohaemagglutinin, irradiated cells and conditioned medium containing interleukin-2. For freshly isolated peripheral blood lymphocytes, the mean frequency of clone forming cells (CFC) was 29.4% and for continuously cultured lymphocytes it was 25.5%. Quantitative considerations, mixing experiments involving HLA-A2 positive and negative cells and replating experiments indicated that growth in most of the positive microwells originated from a single cell which had a very high self-renewal capacity and underwent approximately 15 divisions during the 14 days of culture. The high plating efficiency and sustained growth observed with this technique suggests that it is the method of choice for enumerating clone forming cells or for isolating clones.
利用植物血凝素、经辐照的细胞和含白细胞介素-2的条件培养基，已开发出一种在微孔板中通过有限稀释法克隆淋巴细胞的简单技术。对于新鲜分离的外周血淋巴细胞，克隆形成细胞（CFC）的平均频率为29.4%，而对于连续培养的淋巴细胞，该频率为25.5%。定量分析、涉及HLA-A2阳性和阴性细胞的混合实验以及再铺板实验表明，大多数阳性微孔中的生长源自单个细胞，该细胞具有非常高的自我更新能力，并且在14天的培养过程中经历了约15次分裂。用该技术观察到的高接种效率和持续生长表明，它是计数克隆形成细胞或分离克隆的首选方法。
