Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting.
Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting.
复制标题
DOI：
10.1101/pdb.prot087387
复制
发表时间：
2016-04-01
影响因子：
--
通讯作者：
Meier, Pascal
中科院分区：
文献类型：
--
作者：
Wallberg, Fredrik;Tenev, Tencho;Meier, Pascal
荧光激活细胞分选（FACS）是一种基于激光的生物物理技术，允许同时进行多参数分析。对于垂死细胞的分析，荧光标记的膜联蛋白V（Annexin V（FITC））和碘化丙啶（PI）是最常用的试剂。代替PI，也可以使用4 '，6-二脒基-2-苯基吲哚（DAPI）。DAPI是一种荧光染料，与DNA中富含A-T的区域强烈结合。DAPI和PI只能低效地穿过完整的细胞膜，因此优先染色死细胞。DAPI可以与膜联蛋白V（FITC）和电位荧光染料四甲基罗丹明甲酯（TMRM）组合，其测量线粒体渗透性转变和线粒体膜去极化。TMRM是一种细胞可渗透的荧光染料，被隔离到活性线粒体，因此标记活细胞。在凋亡或坏死性凋亡时，TMRM信号丢失。使用膜联蛋白V（FITC）/DAPI/TMRM的优点是整个细胞群被标记，并且很容易区分活细胞（TMRM + /膜联蛋白V（FITC）-/DAPI-）与垂死或死亡细胞（凋亡：TMRM-/膜联蛋白V（FITC）+ /DAPI-;坏死：TMRM-/膜联蛋白V（FITC）+ /DAPI+）。这一点很重要，因为必须避免细胞碎片（荧光阴性颗粒）以建立用于FACS分析的正确参数，否则将获得不正确的统计值。为了获得样品中细胞浓度或绝对细胞计数的信息，建议向流式细胞仪样品中加入内部微球计数标准品。该方案描述了HT 1080和L929细胞中细胞死亡的FACS分析，但它可以容易地适用于其他感兴趣的细胞类型。
Fluorescence-activated cell sorting (FACS) is a laser-based, biophysical technology that allows simultaneous multiparametric analysis. For the analysis of dying cells, fluorescently labeled Annexin V (Annexin V(FITC)) and propidium iodide (PI) are the most commonly used reagents. Instead of PI, 4',6-diamidino-2-phenylindole (DAPI) can also be used. DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. DAPI can be combined with Annexin V(FITC)and the potentiometric fluorescent dye, tetramethylrhodamine methyl ester (TMRM), which measures mitochondrial permeability transition and mitochondrial membrane depolarization. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells. On apoptosis or necroptosis the TMRM signal is lost. The advantage of using Annexin V(FITC)/DAPI/TMRM is that the entire cell population is labeled, and it is easy to distinguish living (TMRM + /Annexin V(FITC)-/DAPI-) from dying or dead cells (apoptosis: TMRM-/Annexin V(FITC)+ /DAPI-; necrosis: TMRM-/Annexin V(FITC)+ /DAPI+). This is important because cell debris (fluorescent negative particles) must be avoided to establish the correct parameters for the FACS analysis, otherwise incorrect statistical values will be obtained. To obtain information on the cell concentration or absolute cell counts in a sample, it is recommended to add an internal microsphere counting standard to the flow cytrometric sample. This protocol describes the FACS analysis of cell death in HT1080 and L929 cells, but it can be readily adapted to other cell types of interest.