Fluostar optima 96 well plate reader

To evaluate cytotoxic effects of the tested extracts, compounds and diluent (DMSO), cell viability was studied using Cell Counting Kit-8 (CCK8, Merck, Germany). The protocol was the same for CRFK and VeroE6 cells: cells were seeded at 10,000 cells per well of flat bottom 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark). The next day, medium was exchanged containing specified concentrations of the samples. Each concentration or dilution was tested in three replicates; at least six nontreated control wells were included in the assay. After 45 min or 24 h of incubation at 37 °C and 5% CO₂, CCK8 Reagent (10 µL) was added per well and plates were incubated for 1 h at 37 °C, prior to recording absorbance at 450 nm using a FLUOstar OPTIMA 96-well plate reader (BMG LABTECH, Offenburg, Germany). The viability of CRFK cells was only tested after 45 min incubation. Absorbance recorded in each well was related to the average absorbance of nontreated control wells to calculate the percentage of cell viability. 1% SDS was used as negative control. Sigmoidal dose response curves were fitted, and median cytotoxic concentration (CC₅₀) values were calculated with GraphPad Prism 8.0.0.

To evaluate cytotoxic effects of the studied inhibitors, cell viability assays were done as described (15 (link), 26 (link), 27 (link)) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). VeroE6 or A549-hACE2 cells, plated the previous day at 10,000 cells per well in 96-well flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark), were treated with the specified concentrations of inhibitors, including three replicate wells per concentration and 10 nontreated control wells. Following incubation for 46 to 50 hours at 37°C and 5% CO₂, CellTiter 96 AQueous One Solution Reagent was added at 20 μl per well followed by incubation for 1.5 to 2 hours at 37°C and 5% CO₂ and determination of absorbance at 492 nm using a FLUOstar OPTIMA 96-well plate reader (BMG LABTECH, Offenburg, Germany). For calculation of cell viability in %, absorbance values from individual treated wells were related to the mean absorbance of nontreated control wells. Data points are means of triplicates with SEMs. Sigmoidal concentration-response curves were fitted, and 50% cytotoxic concentration values were calculated using GraphPad Prism 8.0.0 with a bottom constraint of 0 applying the equation Y = Top/[1 + 10^{(log10EC50 − X)*HillSlope}].