Synergy htx plate

Cell viability was measured based on quantification of ATP, which serves as an indicator of metabolically active cells using Promega CellTiter-Glo Luminescent Cell Viability Assay (Madison, WI, USA). After the 24-h exposure period, the plate was brought to room temperature and an equal volume of the CellTiter-Glo reagent was added to each well. The plate was then protected from light using foil and placed on an orbital shaker at 10 rpm for 15 min. Full-spectrum luminescence was immediately read using Synergy HTX plate Bio Tek plate reader (Winooski, VT, USA).

Lactate Dehydrogenase (LDH) leakage was measured in cell media as an indicator of cytotoxicity from chemical treatments using Cyquant LDH Kit (Thermo Fischer Scientific, Waltham, MA, USA). Menadione (200 µM), which has shown to induce significant cytotoxicity in our cells, was used as a positive control and cells incubated in cell media, and 2% DMSO served as a vehicle control. After the 24-h exposure period, equal volumes of cell media and LDH reagent were transferred to a fresh 96-well plate and incubated away from light for 30 min. An equal volume of the stop solution was added, and absorbance was read at 490 nm and background at 680 nm using a Synergy HTX plate Bio Tek plate reader (Winooski, VT, USA). Cytotoxicity was calculated by subtracting background from absorbance.

Vero cells were seeded in clear bottom, black well tissue culture plates (Corning). Cells at 80% confluence were treated with different concentrations of the RTK inhibitors and infected with EBOV (strain Mayinga), expressing enhanced-GFP, at a multiplicity of infection of 0.1. Virus growth was assessed by measurement of GFP at different time-points using a BioTek Synergy/HTX plate reader with excitation at 485nm and emission at 516nm. Experiments with replication-competent EBOV were performed in the Biosafety Level 4 facility at the National Microbiology Laboratory at the Public Health Agency of Canada in Winnipeg.

Cell viability was measured using a Promega CellTiter-Glo Luminescent Cell Viability Assay (Madison, WI, USA). Cell viability is typically measured based on quantification of ATP, which serves as an indicator of metabolically active cells. Media with 2% DMSO served as the vehicle control and menadione served as the positive control. After the 24-h exposure period, the plate was brought to room temperature and an equal volume of the CellTiter-Glo reagent was added to the black-walled 96-well plate. The plate was then protected from light and placed on an orbital shaker at 10 rpm for 15 min. Full-spectrum luminescence was then read using a Synergy HTX plate Bio Tek plate reader (Winooski, VT, USA).

The presence of intracellular H₂O_2, a type of reactive oxygen species, was measured using Promega ROS-Glo Assay (Madison, WI, USA). At 18 h post-treatment, 20 µL of H₂O₂ Substrate solution was added to each well and incubated at 37 °C and 5% CO₂ for an additional 6 h. 100 µL of the ROS-Glo Detection Solution was then added to each well and incubated at room temperature for 20 min, and full spectrum luminescence was read using Synergy HTX plate Bio Tek plate reader (Winooski, VT, USA).

Reactive oxygen species were measured using DCFDA (Sigma-Aldrich, St. Louis, MO, USA). Neat stock was purchased and diluted in ACS-grade DMSO. After cells were incubated with target compounds, vehicle control, and positive control for 24 h, the dosing solutions were removed and 20 µM DCFDA diluted with HBSS to 2% DMSO was pipetted onto the cells. The plate was then immediately read using a Synergy HTX plate Bio Tek plate reader (Winooski, VT, USA) at 485/528 excitation and emission.

Strains were inoculated in 5 mL LB supplemented with appropriate antibiotics and grown overnight at 37 °C, 220 RPM. Cultures were then diluted 1:100 in 5 mL EZ-RDM supplemented with antibiotics and grown to OD₆₀₀ 0.5 (using 150 µL samples in flat clear bottomed 96-well plates in a Biotek Synergy HTX plate reader). Cultures were pelleted, flash-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using an Aurum Total RNA Mini Kit (Bio-Rad). 1 µg of RNA was converted to cDNA using iScript reverse transcriptase (Bio-Rad) in 20 µL reactions. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with a 58 °C annealing temperature and 10 µL reaction volumes. qPCR reactions were performed in triplicate on a CFX Connect (Bio-Rad) using 0.5 ng of cDNA, 400 nM primer concentration, and 15 s extension time. 16 S rRNA was used for normalization. Control samples without a template and without reverse transcriptase were analyzed to confirm gene-specific amplification and absence of gDNA contamination. Primer sequences are listed in Supplementary Table 4. Expression levels for each gene were calculated relative to 16 S using the ∆∆C_T method^{64 (link)}.