Darzynkiewicz Z, Bedner E, Traganos F, Murakami T
Brander Cancer Research Institute, New York Medical College, Valhalla, NY 10595, USA.
Hum Cell. 1998 Mar;11(1):3-12.
Despite that large number of methods to analyze cell death, in particular apoptosis, that have been developed, identification of the mode of cell death and its quantitation is not always simple or straightforward. The difficulties, potential pitfalls and traps in quantitation of dead cells, whether apoptotic or necrotic are reviewed. The following are common flaws in the measurement of cell death, which include incorrect assumptions, erroneous data interpretation and shortcomings of the methodology: 1) Misclassification of apoptotic bodies or chromatin fragments as individual apoptotic cells based on cellular DNA content analysis by flow cytometry; 2) Assumption that the quantity of fragmented DNA extracted from cells represents the frequency of apoptosis; 3) Conjecture that the apoptotic index represents the cell death rate; 4) Assumption that apoptotic cells must exhibit classical features of apoptosis e.g. internucleosomal DNA fragmentation; 5) Inadequacy of methods that presume to discriminate between late apoptotic and necrotic cells; 6) Possibility of a selective enrichment or loss of apoptotic cells during cell separation on density gradients, during trypsinization or other procedures of cell collection; and 7) Inability to distinguish between live, nonapoptotic cells phagocytizing apoptotic bodies and genuine apoptotic cells by flow cytometric methods. Many of the problems stem from the difficulty in identifying apoptotic or necrotic cells. Because apoptosis and necrosis have been originally defined based on morphological criteria it is essential to confirm the mode of cell death by microscopy. Laser scanning cytometry (LSC), which combines the advantages of flow and image cytometry, offers the possibility of morphological examination of apoptotic cells. By virtue of this attribute LSC appears to be the instrument of choice for analysis of apoptosis.
尽管已经开发出大量用于分析细胞死亡，尤其是凋亡的方法，但确定细胞死亡的模式及其定量分析并不总是简单直接的。本文综述了定量分析死亡细胞（无论是凋亡还是坏死）时存在的困难、潜在的缺陷和陷阱。以下是细胞死亡测量中常见的缺陷，包括错误的假设、错误的数据解读和方法学的不足：1）基于流式细胞术对细胞DNA含量分析，将凋亡小体或染色质片段误分类为单个凋亡细胞；2）认为从细胞中提取的片段化DNA数量代表凋亡频率的假设；3）推测凋亡指数代表细胞死亡率；4）认为凋亡细胞必须表现出凋亡的经典特征，如核小体间DNA断裂；5）假定用于区分晚期凋亡细胞和坏死细胞的方法不充分；6）在密度梯度离心、胰蛋白酶消化或其他细胞收集过程中，凋亡细胞可能会选择性富集或丢失；7）无法通过流式细胞术方法区分吞噬凋亡小体的活的、非凋亡细胞和真正的凋亡细胞。许多问题源于难以识别凋亡或坏死细胞。由于凋亡和坏死最初是根据形态学标准定义的，因此通过显微镜确认细胞死亡模式至关重要。激光扫描细胞术（LSC）结合了流式细胞术和图像细胞术的优点，为凋亡细胞的形态学检查提供了可能。凭借这一特性，LSC似乎是分析凋亡的首选仪器。
