Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting.
Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting.
复制标题
使用荧光激活细胞分选快速分离克隆的同型转换变体。
DOI：
10.1002/cyto.990020607
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Herzenberg,LA
中科院分区：
文献类型：
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作者：
Dangl,JL;Parks,DR;Oi,VT;Herzenberg,LA
我们使用针对小鼠免疫球蛋白同种型的高度特异性、直接荧光素缀合的异源（传统）和单克隆抗体，结合荧光激活细胞分选仪（FACS）来富集和克隆产生新免疫球蛋白重链恒定区的杂交瘤细胞。每个变体保留亲本重链可变区和亲本免疫球蛋白轻链；因此，每个变体都结合相同的丹酰基 (DNS) 半抗原。这些同种型转换变体发生的频率约为 10−5 至 10−6。我们能够通过首先分选大约 1000 倍富集所需的免疫球蛋白产生细胞，将这些细胞培养 5 到 9 天，然后进行第二次 1000 倍富集并直接将细胞克隆到 96 孔培养托盘中来分离变体。在最初选择后仅 3-5 周就对克隆进行筛选，以分泌所选同种型的丹酰结合免疫球蛋白。现有方法的明智组合允许使用细胞分选仪改进分析技术。这些包括：首先，用溴化乙锭或碘化丙啶对死细胞进行“红色”荧光染色，并使用红色荧光测量将死细胞排除在绿色荧光选择之外；其次，使用荧光信号的对数放大，可以更简洁地选择用于分选的荧光参数。
We have used highly specific, directly fluorescein‐conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10−5to 10−6. We were able to isolate the variants by first sorting for an approximate 1000‐fold enrichment of the desired immunoglobulin‐producing cells, growing these cells for five to nine days, followed by a second 1000‐fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3–5 weeks after the original selection for secretion of dansyl‐binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, “red” fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.