Wst 1

The WST-1 assay, a measure of cell viability based on the reduction of a tetrazolium compound to a soluble derivative was used [36 (link)]. The absorbance recorded at 420 nm is directly proportional to the number of living cells in culture. At 80%-90% confluence, cells were added to 96-well micro-well plates at a density of 5,000 cells/well and incubated overnight. They were then exposed to increasing concentrations of OP or left untreated as controls for 24, 48, and 72 hours. Absorbance readings were taken at 0, 24, 48, and 72 hours by adding WST-1 (Roche Diagnostics Division de Hoffman La Roche Limitée, Laval-des-Rapides, QC, Canada) as a cell proliferation reagent to each well (10% WST-1 in Dulbecco's Modified Eagle's Medium) followed by incubation at 37°C for 2 hours before reading at the indicated time points. Cell viability was presented as a percentage of control, and illustrated as a bar graph using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). The following formula was used to determine cell viability as a percent of control for each time point and OP/chemotherapeutic drug concentration:
[(Absorbance of cells in given concentration of drug) – (Media absorbance)]/[(Absorbance of cells alone) – (Media absorbance)] × 100.

HeLa cells were exposed to heat shock at 45°C for 0, 7.5 and 15 min and recovered at 37°C for 3 h in fresh media. For real-time cell analyzer analysis, 10,000 cells were plated in 96 well E-plate with 200 μL of media and cell growth was monitored by measuring electrical impedance every 15 min under xCELLigence RTCA SP system (Roche Applied Science). WST-1 cell proliferation assay was performed using WST-1 (Roche). HEK293T cells were transfected with Flag or Flag-hArc vector and treated with heat shock at 45°C for 0, 7.5 and 15 min, then cells were dispensed in 96-well microtiter plates and incubated at 37°C with fresh media for 1 day, and cell survival was measured using WST-1 solution. The cleavage of WST-1 to formazan by metabolically active cells was quantified by scanning the plates in a microtiter plate reader at 440 and 620 nm (reference wavelength). The test medium was used as the background control. Three independent sets of experiments that were performed in triplicate were evaluated. The viability of the treated cells was normalized to the untreated control cells.

DemKT1 cells (3 × 10⁴) were plated in 96-well plates (Corning) and incubated overnight. The cells were infected with viruses at an MOI of 0.1 PFU/cell and incubated at 37°C for 1 h. Subsequently, the supernatant was removed, and the cells were washed with PBS and then incubated in RPMI (100 μL; 2% FBS) for various intervals. WST-1 (Roche) (20 μL) diluted to 10% in PBS was added to the cells and the cells were incubated at 37°C for 1 h. Cell viability was calculated by quantifying the cleavage of WST-1 to formazan by cellular succinate-tetrazolium reductase. Formazan was quantitated by measuring absorbance at A₄₅₀nm-A₆₉₀nm using a PowerScanHT (DS Pharma).