Tsangaris G T, Tzortzatou-Stathopoulou F
University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, University of Athens, Aghia Sophia Children's Hospital, Greece.
In Vivo. 1996 Jul-Aug;10(4):435-43.
In vitro and ex vivo many methods are effective for the quantitation of apoptotic cells. These methods cannot be applied for the study of apoptosis in peripheral blood (PB) samples, because of the disappearance of circulating apoptotic cells and the heterogeneity of cell populations. In the present study we describe a nuclear chromatin staining method using ethidium bromide (EtBr), by which we were able to identify and quantify morphologically apoptotic cells in an early stage of the apoptotic process, before their disappearance. The application of the EtBr nuclear stain in CCRF-CEM cells incubated with epirubicin, provides a reliable and reproducible estimation of apoptosis compared with other assays. The main advantage of this method is that it permits the study of four cellular subpopulations by their morphological characteristics (normal cells, early apoptotic, late apoptotic and cells which died from necrosis). Furthermore, we utilized the EtBr nuclear stain to identify and quantify the circulating apoptotic cells directly in the PB of patients with acute lymphoblastic leukemia during multidrug chemotherapy. Circulating apoptotic cells were detectable after 24 hours treatment followed by the decrease of peripheral blood mononuclear cells. Thus staining of nuclei with EtBr provides a rapid and useful method for the quantitative study of apoptotic cells in somatic fluids during clinical trials.
在体外和离体实验中，有许多方法可有效定量凋亡细胞。但由于循环凋亡细胞的消失和细胞群体的异质性，这些方法无法应用于外周血（PB）样本中凋亡的研究。在本研究中，我们描述了一种使用溴化乙锭（EtBr）的核染色质染色方法，通过该方法我们能够在凋亡过程的早期，即凋亡细胞消失之前，从形态上识别和定量凋亡细胞。与其他检测方法相比，将EtBr核染色应用于表柔比星处理的CCRF - CEM细胞，能可靠且可重复地评估凋亡情况。该方法的主要优点是可以根据细胞的形态特征（正常细胞、早期凋亡细胞、晚期凋亡细胞和坏死细胞）研究四个细胞亚群。此外，我们利用EtBr核染色直接在急性淋巴细胞白血病患者多药化疗期间的外周血中识别和定量循环凋亡细胞。治疗24小时后可检测到循环凋亡细胞，随后外周血单个核细胞减少。因此，用EtBr对细胞核进行染色为临床试验期间体液中凋亡细胞的定量研究提供了一种快速且有用的方法。
