自噬检测方法(自噬pc)

导语：实验干货 | 告别转染，自噬检测试剂盒Cyto-ID简介

自噬检测试剂盒Cyto-ID简介

品牌：Enzo

货号：ENZ-KIT175-0050、ENZ-KIT175-0200

产品名称：CYTO-ID® Autophagy detection kit 2.0（Cyto-ID细胞自噬检测试剂盒2.0）

CYTO-ID Autophagy Detection Kit 2.0使用新型染料检测自噬小泡和监控活细胞自噬流，选择性标记积累的自噬小泡。染料已通过优化，不染溶酶体，在自噬前体、自噬体和自噬溶酶体里呈现出明亮的荧光。该试剂盒提供了一种无需细胞转染，可以在活细胞中监控细胞自噬的快速定量方法。

◆ 特点

1) 更明亮，更而耐光，特异性染色自噬小泡

2) 无需转染及转染效率验证

3) 快速量化原生异质性细胞群中的自噬

4) 不染溶酶体，减少其他染料的背景干扰

5) 便于高通量筛选自噬激活剂和抑制剂

◆ 原理

该产品所用探针是阳离子两亲性示踪剂（CAT）染料，以类似诱导磷脂药物的方式迅速进入到细胞中。染料的功能基团能够选择性标记与自噬通路相关的小泡，而不会在溶酶体中聚集。胞内物质被扩大的膜囊包裹，吞噬泡形成双层膜囊泡，成为自噬体。自噬体外膜随后与溶酶体融合，和内部物质被自噬性溶酶体降解。

◆ 使用该试剂盒检测自噬的途径

1、测定吸光度和荧光图谱（499/548nm）来检测自噬

Absorbance and fluorescence emission spectra (499/548 nm) for CYTO-ID® Green Detection Reagent 2 (panel A) were determined in PBS. Absorbance and fluorescence emission spectra (350/461 nm) for Hoechst 33342 (panel B) were determined in 1X Assay Buffer.

2、用激光共聚焦显微镜或荧光显微镜来检测自噬

Cells were stained with CYTO-ID® Green Detection Reagent 2 after being administrated. Autophagic cells showe very bright green fluorescent signals and punctate structures.

3、运用流式细胞仪来分析细胞自噬的产生

Flow cytometry-based profiling of autophagy in cells. After staining with CYTO-ID® Green Detection Reagent 2 for 30 minutes, cells were washed and analyzed by flow cytometry. Autophagic cells show an increase in fluorescence.

4、微孔板法分析细胞自噬的产生

Microplate-based profiling of autophagy in cells. The autophagy cells have an increase values in microplate detection.

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