Listman J A, Wang Y, Castro J E, Rimm I J, Finn P W, Perkins D L
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Cytometry. 1998 Nov 1;33(3):340-7.
The flow cytometric analysis of apoptosis in lymphocytes from in vivo samples has been difficult because of the low frequency of apoptotic events. To overcome this obstacle, many investigators have relied on in vitro incubations to increase the number of apoptotic cells before analysis. In this report, we show that an adaptation of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay for use in flow cytometry can be used to detect rare apoptotic lymphocytes from freshly harvested LN suspensions. This approach is both specific and extremely sensitive. This method also is amenable to multiparameter analyses and allows a phenotypic analysis of these rare apoptotic cells. However, we observed that some monoclonal antibodies can stain apoptotic-but not viable-cells nonspecifically. Therefore, the specificity of all antibodies to stain apoptotic cells was confirmed in competition assays.
由于体内样本淋巴细胞凋亡事件的频率较低，对其进行流式细胞术分析一直颇具难度。为克服这一障碍，许多研究者依靠体外孵育来增加分析前凋亡细胞的数量。在本报告中，我们表明，一种适用于流式细胞术的末端脱氧核苷酸转移酶（TdT）介导的dUTP-地高辛配基缺口末端标记（TUNEL）检测方法，可用于检测新鲜采集的淋巴结悬浮液中罕见的凋亡淋巴细胞。这种方法既特异又极其灵敏。该方法也适用于多参数分析，并能对这些罕见的凋亡细胞进行表型分析。然而，我们观察到一些单克隆抗体可非特异性地染色凋亡细胞而非活细胞。因此，在竞争试验中证实了所有抗体对凋亡细胞染色的特异性。
