Cell viability staining for flow cytometry
Assessment of the cell viability within the flow cytometry experiments is necessary for proper setup and analysis. Dead cells may cause various problems for evaluation of the experiment, mainly aggregation and nonspecific binding of detection antibodies. Determination of dead cell is also a crucial step for cell sorting and their appropriate labeling helps to exclude them, especially when the viable cells are used for further experiments. Dead cells undergo morphological or physiological changes. Viability dyes for flow cytometry are based on the increased permeability of the cell membrane or decreased enzymatic activity of dead cells.
DNA staining of dead cells Group of DNA binding dyes that are not passing through intact cell membrane or that are cell permeable, but living cells transport them immediately out of the cell. Thus only DNA of dead cells is stained. DNA binding dyes are easy to use and quick. They are not intended for fixed cells viability staining, but may be used for cell cycle staining of fixed cells. Propidium Iodide, 7-AAD, DAPI, Hoechst, Ethidium bromide, commercial - Sytox, TO-PRO, DRAQ Amine reactive dyes
Dyes that are able to react with amine groups of proteins. Live cells with intact cell membrane are stained only on the surface, dead cells are stained also intracellularly and provide 50-100x higher signal intensity. Cells stained with amine reactive dyes may be fixed. Several fluorochroms available, suitable for combinations with wider cytometry panels. Commercial names – Live/dead, Zombie, Ghost, Horizon, Vivafix Staining of viable cells These dyes are changed from a non fluorescent to fluorescent state by enzymes, that are active only in living cells. Fluorescent signal from dead cell is much lower than from viable cells. Dyes are usually permanent and do not affect cell functions. They are not fixable, but may be used also for proliferation assays. CFSE, 5-CFDA, Resazurin, commercial – Cell tracker
References
1. Simon Johnson, Vy Nguyen, and David Coder, Assessment of Cell Viability, Current Protocols in Cytometry 9.2.1-9.2.26, April 2013
2. Kummrow A, Frankowski M, Bock N, Werner C, Dziekan T, Neukammer J. Quantitative assessment of cell viability based on flow cytometry and microscopy. Cytometry A. 2013 Feb;83(2):197-204. doi: 10.1002/cyto.a.22213. Epub 2012 Oct 18. PMID: 23081720.
3. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M. Amine-reactive dyes for dead cell discrimination in fixed samples. Curr Protoc Cytom. 2010 Jul;Chapter 9:Unit 9.34. doi: 10.1002/0471142956.cy0934s53. PMID: 20578108; PMCID: PMC2915540.