Matteucci C, Grelli S, De Smaele E, Fontana C, Mastino A
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
Cytometry. 1999 Feb 1;35(2):145-53. doi: 10.1002/(sici)1097-0320(19990201)35:2<145::aid-cyto6>3.0.co;2-2.
Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells.
Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide.
Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining.
A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.
广泛用于检测细胞凋亡的方法无法让我们轻易区分活细胞与坏死细胞核。即使在单细胞水平上凋亡和坏死似乎是相互替代发生的，但它们在细胞群体中同时出现的频率可能比预期的要高得多。因此，人们将注意力集中在试图通过多参数流式细胞术识别活细胞以及凋亡或坏死死细胞的特征上。
分别使用地塞米松或前列腺素E2处理以及60℃热休克或过氧化氢，在体外诱导小鼠胸腺细胞和成年外周血淋巴细胞发生凋亡和坏死。将传统方法，如DNA凝胶电泳、碘化丙啶染色后进行单荧光分析或使用流式细胞术进行膜联蛋白-V-异硫氰酸荧光素加碘化丙啶染色，与一种新方法进行比较。该新方法包括通过低渗溶液分离细胞核、高剂量去污剂处理以及用碘化丙啶进行DNA染色后，通过流式细胞术进行联合光散射和红色荧光分析。
结果表明，尽管DNA凝胶电泳和单参数荧光流式细胞术分析等传统方法正如预期的那样无法区分细胞活力、凋亡和坏死，但我们的新方法使我们能够轻松识别活细胞、凋亡细胞和坏死细胞的细胞核。使用我们的方法获得的结果与使用碘化丙啶/膜联蛋白V染色后对细胞进行双色分析获得的结果相当。
已开发出一种高度可重复、廉价、快速且易于操作的分析方法，用于同时检测细胞凋亡和坏死。
