Glisic-Milosavljevic S, Waukau J, Jana S, Jailwala P, Rovensky J, Ghosh S
Max McGee National Research Center for Juvenile Diabetes and Human Molecular and Genetics Center, Medical College and Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA.
Cell Prolif. 2005 Oct;38(5):301-11. doi: 10.1111/j.1365-2184.2005.00351.x.
Death through apoptosis is the main process by which aged cells that have lost their function are eliminated. Apoptotic cells are usually detected microscopically by changes in their morphology. However, determination of early apoptotic events is important for in vitro (and ex vivo) studies. The main objective of the present study is to find the most sensitive method for apoptosis detection in human peripheral blood mononuclear cells (PBMCs) by comparing six different methods following five different means of immunological stimulation at 3 and 5 days. Each of six apoptosis quantification methods, except the trypan blue exclusion test, is a combination of two stains, one for the specific detection of apoptotic cells and the other for the unspecific detection of dead cells. Values for apoptosis and mortality were compared with a reference method. The choice of apoptosis detection method is more important following 3 days of stimulation than after 5 days of stimulation (P=2x10(-6) versus P=1x10(-2)). In contrast, we find mortality measurements following the different means of stimulation highly significant at both 3 and 5 days (F2.28=7.9, P=1.4x10(-6) at 3 days and F2.28=8.5, P=4.5x10(-7) at 5 days). Variation as a result of the combination of specific PBMC stimulation and the method used to detect apoptosis is reduced considerably with time (F1.58+3.7, P+3x10(-7) at 3 days to F=1.58=0.97, P=0.5 at 5 days). Based on Tukey's test, YO-PRO-1 is the most sensitive stain for apoptosis and, when combined with 7-AAD, provides an accurate measure of apoptosis and mortality. In conclusion, we propose YO-PRO-1/7-AAD as a new combination and low-cost alternative for the sensitive detection of early apoptosis.
通过凋亡导致的细胞死亡是清除失去功能的衰老细胞的主要过程。凋亡细胞通常通过显微镜下形态学的变化来检测。然而，对于体外（和离体）研究而言，确定早期凋亡事件很重要。本研究的主要目的是通过比较六种不同方法，在5天和3天时采用五种不同的免疫刺激方式，找出检测人外周血单个核细胞（PBMC）凋亡的最敏感方法。六种凋亡定量方法中，除台盼蓝排斥试验外，每种方法都是两种染色剂的组合，一种用于特异性检测凋亡细胞，另一种用于非特异性检测死亡细胞。将凋亡和死亡率的值与参考方法进行比较。刺激3天后选择凋亡检测方法比刺激5天后更重要（P = 2×10⁻⁶ 对比 P = 1×10⁻²）。相反，我们发现在3天和5天时，不同刺激方式下的死亡率测量结果均具有高度显著性（3天时F2.28 = 7.9，P = 1.4×10⁻⁶；5天时F2.28 = 8.5，P = 4.5×10⁻⁷）。随着时间推移，特异性PBMC刺激与用于检测凋亡的方法相结合所导致的差异显著降低（3天时F1.58 + 3.7，P + 3×10⁻⁷；5天时F = 1.58 = 0.97，P = 0.5）。基于Tukey检验，YO-PRO-1是检测凋亡最敏感的染色剂，与7-AAD联合使用时，可准确测量凋亡和死亡率。总之，我们提出YO-PRO-1/7-AAD作为一种新型组合且低成本的替代方法，用于早期凋亡的灵敏检测。
