细胞凋亡检测试剂盒——Annexin V, FITC Apo

细胞凋亡是指为维持有机体内环境稳定，由基因控制的细胞自主的有序的死亡。正常情况下任何细胞在形成过程中发生的异常都会通过凋亡消除。例如体内的癌细胞增长为肿瘤的过程会受细胞凋亡的引导而被抑制。然而在抑癌基因p53出现问题时，凋亡就不会诱导发生，从而导致癌细胞的不断增长。细胞凋亡可以通过细胞形态的变化或生物化学的变化来检测。目前常用的指标有caspase活性变化、DNA碎片、磷脂酰丝氨酸的外翻等。

Annexin V染色的细胞可以用于检测细胞凋亡早期的细胞膜变化。在细胞凋亡早期，膜磷脂酰丝氨酸由脂膜内侧翻向外侧。Annexin V是一种分子量为35-36kD的Ca²⁺依赖性磷脂结合蛋白，与磷脂酰丝氨酸有高度亲和力，可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜特异性结合，因此Annexin V 被作为检测细胞早期凋亡的灵敏指标之一。用绿色荧光FITC标记的Annexin V通过流式细胞仪或荧光显微镜可以检测到细胞凋亡的发生。碘化丙啶 (Propidium Iodide, PI) 是一种核酸染料，PI只能透过凋亡晚期和死细胞的细胞膜，因此Annexin V和PI结合使用，可以区分凋亡早晚期的细胞及死细胞。

                                                         Normal Cell                         Early stages of apoptosis                   Late stages of apoptosis

-流式细胞仪检测

1. 加样到流式细胞仪检测，激发波长Ex = 488 nm；发射波长Em = 530 nm。

2. Annexin V-FITC的绿色荧光通过FITC通道 (FL1) 检测；PI的红色荧光通过PI 通道 (FL2或FL3) 检测 (建议使用FL3)。

-荧光显微镜检测

 将细胞悬液滴到玻片上，置于显微镜上使用合适的滤光片检测。* 由于EDTA会对细胞膜的特定部位有损伤，建议使用不含EDTA的胰酶消化贴壁细胞。

所需的设备和材料

   - 样品和诱导剂

   - PBS、超纯水

   - 合适量程的移液器

   - 细胞培养用6，12，24，96孔板

   - 流式细胞仪或荧光显微镜。

   * 最大激发/发射波长  Annexin V, FITC: 494 nm/518 nm；PI: 535 nm/617 nm

溶液制备

   1×Annexin V Binding solution：将10×Annexin V Binding Buffer用超纯水稀释10倍。

稳定性

   试剂盒在0-5℃下可以保存6个月，请避光保存Annexin V, FITC结合物。

＊实验实例及操作步骤请参考中文说明书

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