Toba K, Winton E F, Bray R A
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
Cytometry. 1992;13(1):60-7. doi: 10.1002/cyto.990130110.
We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.
我们开发了一种改进的三重染色技术，该技术允许同时进行细胞表面抗原的流式细胞荧光分析、溴脱氧尿苷掺入DNA以及使用7-氨基放线菌素D进行DNA定量。将PHA激活的人外周血淋巴细胞与溴脱氧尿苷一起孵育，并用藻红蛋白标记的单克隆抗体对细胞表面表型进行染色。染色后的细胞先用1%多聚甲醛和45%乙醇依次固定。固定后的细胞先用抗BrdUrd单克隆抗体染色，然后用异硫氰酸荧光素（FITC）偶联的山羊抗小鼠抗体染色，并与7-氨基放线菌素D孵育。固定后所有步骤均使用低渗缓冲液。通过流式细胞荧光术对染色固定的细胞进行分析，以同时检测绿色（525nm）、橙色（570nm）和红色（大于650nm）荧光。利用这种染色技术，我们能够通过单激光激发同时分析细胞表型、DNA合成和总细胞DNA含量。
