Streamlined duplex live-dead microplate assay for cultured cells. (August 2017)

• Record Type: Journal Article
• Authors: Pfeffer, Bruce A.; Fliesler, Steven J.
• Is Part Of: Experimental eye research. Volume 161(2017)
• Page Start: 17
• Page End: 29
• Publication Date: 2017-08
• Subjects: Cell viability assay -- Calcein AM -- Sytox Orange -- Retinal cell line -- Fluorescence assay -- Plate reader; 7kCHOL 7-ketocholesterol -- ABCB1 ATP-binding cassette sub-family B member 1 (P-glycoprotein) -- ABCC1 ATP-binding cassette sub-family C member 1 (Multidrug resistance-associated protein 1) -- ANOVA analysis of variance -- Ar argon -- BSA bovine serum albumin -- CaAM calcein acetoxymethyl ester -- di deionized -- DMSO dimethyl sulfoxide -- HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid -- IM incubation medium -- MEBSS modified Earle's balanced salt solution -- MHBSS modified Hanks' balanced salt solution -- PORN poly-L-ornithine -- PPE personal protective equipment -- RFU relative fluorescence units -- rt room temperature -- SE standard error -- SO SYTOX® Orange -- Stsp staurosporine -- TCP tissue culture plastic -- VC vehicle control; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2017.05.011
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: A duplex fluorescence assay to assess the viability of cells cultured in multi-well plates is described, which can be carried out in the original culture plate using a plate reader, without exchanges of culture or assay medium, or transfer of cells or cell supernatant. The method uses freshly prepared reagents and does not rely on a proprietary, commercially supplied kit. Following experimental treatment, calcein acetoxymethyl ester (CaAM) is added to each well of cultured cells; after 30 min, the fluorescence intensity (emission λ max ∼ 530 nm) is measured. The signal is due to formation of calcein, which is produced from CaAM by action of esterase activity found in intact live cells. Since live cells may express plasma membrane multidrug transport proteins, especially of the ABC transporter family, the CaAM incubation is carried out in the presence of an inhibitor of this efflux process, thereby improving the dynamic range of the assay. Next, SYTOX ® Orange (SO) is added to the culture wells, and, after a 30-min incubation, fluorescence intensity (emission λ max ∼ 590 nm) is measured again. SO is excluded from cells that have an intact plasma membrane, but penetrates dead/dying cells and can diffuse into the nucleus, where it binds to and forms a fluorescent complex with DNA. The CaAM already added to the wells causes no interference with the latter fluorescent signal. At the conclusion of the duplex assay, both live and dead cells remain in the culture wells and can be documented by digital imaging to demonstrate correlation of cellular morphology with the assay output. Two examples of the application of this method are provided, using cytotoxic compounds having different mechanisms of action. Graphical abstract: Highlights: Rapid, duplex, single well cell viability assay is performed without media changes, or transfer of cells or cell supernatant. After treatments, live cells detected by enzymatic hydrolysis of CaAM ester, with calcein as the fluorescent product. Next Sytox Orange detects dead, permeable cells, based on fluorescence of complex with DNA, with no calcein interference. Inhibitor of multidrug transport included to inhibit efflux of calcein, improving the dynamic range of the assay. Detailed examples of the protocol using different cytotoxic compounds tested on two retinal cell lines are provided.