Streamlined duplex live-dead microplate assay for cultured cells

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 λmax ∼ 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 λmax ∼ 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.

Transcriptomic Changes Associated with Loss of Cell Viability Induced by Oxysterol Treatment of a Retinal Photoreceptor-Derived Cell Line: An In Vitro Model of Smith–Lemli–Opitz Syndrome

“…661W cells are a simian virus 40 (SV40) large T antigen-immortalized cell line derived from a benign, induced retinal tumor isolated from an early postnatal mouse, and had been originally demonstrated to possess properties of cone photoreceptors [ 100 , 226 ]. Protocols and techniques for expanding initial 661W cultures and cryopreserving source stocks, for subculturing and adaptation to a more defined medium, and for the extensive characterization and authentication of these cells in our laboratory using genomic, transcriptomic, immunochemical/immuno-histochemical, and morphological analyses and criteria, have been previously provided in detail [ 21 , 242 ], and are briefly summarized here. Cells were received in passage 24, and underwent gradual adaptation during the next several passages from medium containing 10% fetal bovine serum (Atlanta Biological, Atlanta, GA) to growth medium with the following attributes (see also Table 3 in [ 242 ]): a greatly reduced serum component (0.2% ( v / v ) bovine calf serum (BCS; Lonza, Walkersville, MD, USA)); formulation using a basal medium composed of 1:1 DMEM:F-12 (both HEPES modification; Sigma-Aldrich); addition of supplements based on an updated recipe for Neurobasal medium [ 243 , 244 ]; and containing a HEPES-buffered saline extract of a bovine retinal homogenate as the only other undefined component, contributing added total protein for a final concentration of 6 mg per liter of complete medium.…”

“…Cultures underwent routine incubation at 36.5 °C, in a 6% CO 2 /90% humidified air. After 2 days, when the cultures had attained approximately 70% confluence, growth medium was replaced with 9 mL of incubation medium [ 21 ], a simplified medium composed of DMEM/F-12 with only the following substituents: BCS (0.2%), transferrin, hydrocortisone, non-essential amino acids, alanyl-glutamine, sodium pyruvate, triiodothyronine, glucose, fructose, and 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO) (Sources available in [ 242 ]). After overnight incubation in incubation medium, 1 mL per dish of one of the 10× working stocks of treatment agents was introduced, each dish was briefly swirled to effect mixing, and the dishes were incubated for time intervals noted below subsequent to harvesting of RNA.…”

“…Chamberslides (4-well), made with surface-modified glass as per Kleinfield et al [ 252 ] (Lab-Tek System II, Thermo Fisher Scientific, Waltham, MA), were first treated with poly-L-ornithine (4 µg/cm 2 ; working stock in sterile water, diluted from 0.01% ( w / v ) source stock obtained from Sigma-Aldrich) [ 242 ], and then 661W cells between passages 40 and 50 were seeded at 10,000 cells/well, arranged in four treatment designations: EPCD (6, 8, or 10 µM); DMSO VC (0.1 % ( v / v ), matching the final dilution from EPCD stock); 7kCHOL (20 or 25 µM); and hpβCD VC (0.009% ( w / v )), matching the lower dilution from 7kCHOL working stock). Stocks and dilutions to 10× desired final concentrations of experimental agents were created as for the gene array samples, above.…”

Cellulose-Chitosan-Nanohydroxyapatite Hybrid Composites by One-Pot Synthesis for Biomedical Applications

“…The propidium iodide penetrate to cells that have damage in their membranes, and dead cells are detected by the fluorescence produced by the binding of propidium iodide to DNA. Calcein is metabolized in live mitochondria cells by their esterase activity, inducing a green signal [33,34].…”

Microfluidic and Microscale Assays to Examine Regenerative Strategies in the Neuro Retina

“…Contemporary 2D cell culture is, thus, now readily performed in microchambers, where microfluidic volumes are enclosed directly upon a microscope slide to enable the growth of cell groups on the slide itself. Large scale parallelization of microchambers yielded multi-well and microtiter plates, where hundreds to thousands of (sub) microliter compartments, or wells, are fabricated in a uniform footprint on a single substrate [ 167 ]. It is noted that although the substrate length of conventional multi-well plates is often several millimeters long, the characteristic length of individual, parallelized wells is well within the μm range while the wells themselves encompass fluidic volumes of 1 μL or less.…”