Perfetto Stephen P, Chattopadhyay Pratip K, Lamoreaux Laurie, Nguyen Richard, Ambrozak David, Koup Richard A, Roederer Mario
Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.
Curr Protoc Cytom. 2010 Jul;Chapter 9:Unit 9.34. doi: 10.1002/0471142956.cy0934s53.
Amine-reactive dyes, also known as LIVE/DEAD fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm. Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining. Notably, the reaction is irreversible; therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Since amine-reactive dyes are fluorescent when excited by lasers, dead cells can be identified by flow cytometry. This unit describes procedures, troubleshooting, and outcomes for using the two most commonly used amine-reactive dyes, ViViD and Aqua Blue.
胺反应性染料,也称为LIVE/DEAD可固定死细胞染料,是一类适用于鉴定将要固定的样本中死细胞的活力染料。这些染料穿过死细胞的细胞膜,并与细胞质中的游离胺发生反应。活细胞会排斥这些染料,因为它们的细胞膜是完整的,染色后游离染料会被洗去。值得注意的是,这种反应是不可逆的;因此,当细胞被固定并通透化处理时(如细胞内染色程序),结合的染料仍与死细胞相关联(与其他活力染料不同)。由于胺反应性染料在激光激发下会发出荧光,因此可以通过流式细胞术识别死细胞。本单元描述了使用两种最常用的胺反应性染料ViViD和水蓝的程序、故障排除方法及结果。