Dangl J L, Parks D R, Oi V T, Herzenberg L A
Cytometry. 1982 May;2(6):395-401. doi: 10.1002/cyto.990020607.
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 10-5 to 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.
我们使用了高度特异性的、直接用荧光素偶联的异源（传统）抗体和针对小鼠免疫球蛋白同种型的单克隆抗体，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磺酰（DNS）半抗原。这些同种型转换变体的出现频率约为10^-5至10^-6。我们能够通过首先对所需产生免疫球蛋白的细胞进行约1000倍的富集分选，使这些细胞生长5至9天，然后进行第二次1000倍的富集并直接将细胞克隆到96孔培养板中来分离变体。仅在最初选择后3至5周筛选克隆，以检测所选同种型的丹磺酰结合免疫球蛋白的分泌情况。明智地组合现有方法可改进使用细胞分选仪的分析技术。这些技术包括：第一，用溴化乙锭或碘化丙啶对死细胞进行“红色”荧光染色，并利用红色荧光测量从绿色荧光选择中排除死细胞；第二，使用荧光信号的对数放大，以便更简洁地选择用于分选的荧光参数。
