流式分析技术viability-staining.pdfVIP

Flow Cytometry – BestProtocols® Page 1 of 3 Viability Staining Research Use Only  Protocol A: Staining Dead Cells with Propidium Iodide or 7-AAD  Protocol B: Staining Live Cells with Calcein Dyes  Protocol C: Staining Dead Cells with eBioscience Fixable Viability Dyes Introduction Viability staining is an essential component of any flow cytometry experiment. Dead cells can compromise the integrity of the data by non-specifically binding antibodies; therefore it is essential that dead cells be excluded from analysis. eBiosicence® offers several different dyes that can be used to discriminate live and dead cells. Please visit the eBioscience® Technical Support website for more information to help determine which dyes are suitable for the specific application of interest. Protocol A: Staining Dead Cells with Propidium Iodide or 7-AAD Propidium Iodide and 7-AAD can be used to stain dead cells so that they may be excluded from analysis in standard cell surface staining protocols. These dyes cannot pass through intact cell membranes, but may freely enter cells with compromised cell membranes. Upon entering dead cells, Propidium Iodide will intercalate into double- stranded DNA or double-stranded RNA in a stoichiometric manner, while 7-AAD will intercalate only with double- stranded DNA. Because this intercalation is mediated by non-covalent forces, these dyes must remain present in the buffer used to resuspend cells for data acquisition so that dead cells will remain labeled. Reagents  Propidium Iodide Staining Solution (cat. 00-6990)  7-AAD Viability Staining Solution (cat. 00-6993) Experimental Procedure 1. After staining cells for surface antigens, wash cells 1-2 times with flow cytometry staining buffer 2. Resuspend cells in an appropriate volume of flow cytometry staining