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Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry

机译：流式细胞术与AO / EB双重染色检测骨肉瘤细胞凋亡

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Background The aim of this study was to evaluate the ability of dual acridine orange/ethidium bromide (AO/EB) staining to detect tumor cell apoptosis. According to apoptosis-associated changes of cell membranes during the process of apoptosis, a clear distinction is made between normal cells, early and late apoptotic cells, and necrotic cells. Material and Methods We cultured human osteosarcoma cells with 30, 60, and 120 μg/ml kappa-selenocarrageenan. To assess the rates of cell proliferation and apoptosis, cells were fluorescently stained with acridine orange/ethidium bromide (AO/EB) or stained with propidium iodide (PI) and analyzed by flow cytometry. All experiments were repeated at least 3 times. Results Normal tumor cells, early and late apoptotic cells, and necrotic cells were examined using fluorescent microscopy. Early-stage apoptotic cells were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining. Late-stage apoptotic cells were marked with concentrated and asymmetrically localized orange nuclear ethidium bromide staining. Necrotic cells increased in volume and showed uneven orange-red fluorescence at their periphery. Cells appeared to be in the process of disintegrating. The percentage of apoptotic osteosarcoma cells detected by dual acridine orange/ethidium bromide (AO/EB) staining was not significantly different from that detected using flow cytometry (P>0.05). Conclusions Our results suggest that dual acridine orange/ethidium bromide staining is an economic and convenient method to detect apoptosis in tumor cells and to test tumor chemosensitivity compared with flow cytometry.
机译：背景技术这项研究的目的是评估orange啶橙/溴化乙锭（AO / EB）双重染色检测肿瘤细胞凋亡的能力。根据凋亡过程中与细胞凋亡相关的细胞膜变化，在正常细胞，早期和晚期凋亡细胞以及坏死细胞之间有明显的区别。材料和方法我们用30、60和120μg/ ml的Kappa-硒代角叉菜胶培养人骨肉瘤细胞。为了评估细胞增殖和凋亡的速率，将细胞用a啶橙/溴化乙锭（AO / EB）进行荧光染色，或将碘化丙啶（PI）染色，然后通过流式细胞仪进行分析。重复所有实验至少3次。结果使用荧光显微镜检查了正常的肿瘤细胞，早期和晚期凋亡细胞以及坏死细胞。早期凋亡细胞的特征是新月形或颗粒状的黄绿色a啶橙核染色。晚期凋亡细胞用浓缩且不对称定位的橙色核溴化乙锭染色标记。坏死细胞的体积增加并且在其外周显示不均匀的橙红色荧光。细胞似乎正在分解。 a啶橙/溴化乙锭（AO / EB）双染色法检测到的凋亡性骨肉瘤细胞百分比与流式细胞仪检测无显着差异（P> 0.05）。结论我们的结果表明，与流式细胞术相比，dual啶橙/溴化乙锭双染色是一种经济便捷的方法，可检测肿瘤细胞的凋亡并检测肿瘤的化学敏感性。

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