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nm),将人类细胞区分为绿色,以便通过荧光显微镜进行检测。吖啶橙基与dsDNA(Ex=488,Em=520-524)结合时发出绿色荧光,与ssDNA(Ex=457,Em=630-644)或ssRNA(Ex=457,Em=630-444)结合时发红色荧光,也可用于细胞周期分析[1][2][3]。",{"key":242,"value":243},"相关类别","研究领域\n>>\n其他\n信号通路\n>>\n其他\n>>\n其他",{"key":245,"value":246},"体外研究","指南(以下是我们推荐的方案。该方案仅提供指南,应根据您的具体需要进行修改)。1.未固定细胞的DNA和RNA的差异染色[1]：(1)使用发射滤光片和分色镜,在488nm处设置激发流式细胞仪,区分绿色荧光(在515-545nm处测量)和红色发光(最好在640或650nm以上测量)。(2) 将0.2-mL等分的原始细胞悬浮液转移到小玻璃或塑料管中(例如,2-mL或5-mL体积)。在冰上冷却。(3) 轻轻加入0.4 mL冰冷电池渗透溶液。等待15秒,使电池保持在冰上。(4) 轻轻加入1.2 mL冰镇吖啶橙染色液。在黑暗中将电池放在冰上。(5) 添加吖啶橙碱性染色液后2至10分钟内,在流式细胞仪中测量并记录细胞荧光。2.固定细胞的差异染色[1]：(1a)对于悬浮培养或血液样本中的细胞：用冰凉PBS冲洗细胞一次,然后在∼106个细胞\u002FmL。(1b)对于附着在组织培养板上的细胞：用胰酶消化法从烧瓶或培养板中收集细胞,将胰酶消化细胞与悬浮在培养基中的细胞(主要是分离的有丝分裂细胞和死细胞)混合,并用含有血清的培养基冲洗一次,使胰酶失活。将电池悬挂在冰冷的PBS中∼106个细胞\u002FmL。(1c)对于从实体肿瘤中分离出来的细胞：冲洗不含任何用于细胞分离的酶的细胞,并将其悬浮在冰冷的PBS中∼106个细胞\u002FmL。(2) 用巴斯德移液管将1mL细胞悬液转移到含有10mL冰凉70%乙醇的15mL锥形玻璃管中。固定单元格≥冰上2小时。(3) 离心管在300×g,4℃下5分钟。去除所有乙醇,用冰凉PBS冲洗细胞一次,并以\u003C2×106个细胞\u002FmL的密度悬浮在冰凉的PBS中。(4) 提取0.2 mL细胞悬液(≤ 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Differential staining of DNA and RNA. Curr Protoc Cytom. 2004 Nov;Chapter 7:Unit 7.3.\n[2].\nMirrett S. Acridine orange stain. Infect Control. 1982 May-Jun;3(3):250-2.\n[3].\nYektaeian N, et al. Lipophilic tracer Dil and fluorescence labeling of acridine orange used for Leishmania major tracing in the fibroblast cells. 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