Spectramax microplate reader

For serum-binding studies, SARS-CoV-2 S-2P protein was diluted to 5 μg/ml in PBS (pH 7.4) and used to coat 96-well high-binding polystyrene ELISA plates (Corning, Cat# 3690) were coated with 25 μl per well of SARS-CoV-2 S-2P protein diluted to 5 μg/ml in PBS (pH 7.4) and incubated overnight at 4 °C. All subsequent incubations, until the addition of sucbstrate, were for 1-h at 37 °C. Wells were washed three times with PBS and incubated with blocking solution (5% (w/v) non-fat dried milk (NFDM)). Serial dilutions of human serum were prepared in 5% NFDM-PBS and added to the wells (25 μl per well) after the removal of the blocking solution. Plates were washed three times with PBS followed by the addition of secondary cross-adsorbed anti-human IgG-HRP (Thermo Fisher Scientific, Cat# 31413) detection antibody (25 μl per well) at 1:8000 dilution in 5% NFDM-PBS. Plates were washed three times with PBS. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Cat# 34029) was added (25 μl per well) to detect binding, incubated at room temperature for 6–8-min followed by the addition of an equal volume of stop reagent (2 M sulfuric acid). Absorbance was measured at 450 nm using a Spectramax microplate Reader (Molecular Devices), and responses plotted as a function of dilution. Serum binding was calculated as the area under the curve using GraphPad Prism (version 9).

To measure ΔΨ_m in cultured cells, H9c2 cells were incubated with ΔΨ_m-sensitive dye JC-1 (5,5′,6,6′-tetraethyl-benzimidazolylcarbocyanine iodide; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were incubated for 30 min at 37 °C with JC-1 and fluorescence was measured using a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA). J-aggregates (red) and JC-1 dye monomers (green) were monitored at 530 and 590 nm emission (with excitation at 488 nm), respectively. Data are presented as the ratio red/green fluorescence.
ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche, Indianapolis, IN, USA), according to the manufacturer’s recommendations. Luminescence data were normalized to total protein levels.

Enzymatic activity of ETC complexes was determined as previously described [15 (link)], with minor modifications and normalized to mg of mitochondrial protein. All assays were performed at the SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA) at 37 °C.

Total ROS and mtROS production were measured with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and MitoSOX Red, respectively [15 (link)]. Briefly, cells were incubated for 30 min with 10 µM H₂DCFDA or 1 µM of MitoSOX and fluorescence intensity was monitored on the SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA) at the excitation/emission of 599 nm/522 nm (for H₂DCFDA) and 510 nm/580 nm (for MitoSOX).

Crystal violet assays were performed 10 days following drug treatments with either DMSO, trametinib, osimertinib, or vemurafenib. CellTiter-Glo (Promega) experiments were performed as per manufacturer’s protocol. Briefly, cells were plated in a 96-well plate, treated with indicated drug or control and analyzed on a Spectramax microplate reader (Molecular Devices) after 6 days of treatment. Each assay consisted of at least three replicate wells.