Crowley Lisa C, Marfell Brooke J, Waterhouse Nigel J
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia;
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia; Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia; School of Medicine, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.prot087205. doi: 10.1101/pdb.prot087205.
The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes.
健康细胞的细胞核通常呈球形，且DNA均匀分布。在细胞凋亡过程中，DNA会浓缩，但坏死过程中不会发生这种情况。因此，核浓缩可用于区分凋亡细胞与健康细胞或坏死细胞。能与DNA结合的染料，如Hoechst 33342或4',6-二脒基-2-苯基吲哚（DAPI），可用于观察核浓缩。这些染料在紫外光激发下于461 nm处发出荧光，因此可使用配备发射约350 nm光的光源和允许约460 nm光透过的滤光片组的传统荧光显微镜进行观察。本方案描述了用Hoechst 33342染色的细胞的染色和观察方法，但也可适用于用DAPI或其他染料染色。
