I3x plate reader

Fluorescence anisotropy measurements were made in opaque, round-bottom 96-well polystyrene plates using a Molecular Devices i3x plate reader with the Fluorescein-FP cartridge. N-terminally fluorescein-labeled MDM2 was prepared by treating MDM2 with 1 equivalent of NHS-fluorescein (Thermo Fisher Scientific) in 20 mM tris-buffered 150 mM saline + 1 mM TCEP (TBST) at pH 7.2 and incubating overnight at 4 °C before desalting (DoL ~ 0.5). Experiments were conducted using 10 nM fluor–MDM2 and performed in TBST at pH 7.2 plus the indicated inhibitor. Full-length p53 was desalted into the same buffer, and two-fold serial-dilutions were prepared in the 96-well plate. Experimental measurements of parallel and perpendicular fluorescence were blank subtracted using a pair of matched dilution series prepared without fluor–MDM2 to correct for background fluorescence, then converted to anisotropy. The results were plotted versus p53 concentration and fit to a one-site binding equation. K_D and standard error values were determined by inverse-variance weighted pooling of the fit parameters from two replicates.

The trimeric spike protein of Wild, Delta, C.1.2, Omicron variants were coated on high binding 96 well plate (Biomat, MT01F4-HB8) at 0.1 μg per well using phosphate-buffered saline (PBS, pH-7.4) overnight at 4 °C. Blocked with 3% BSA in PBS-T for 2 h at room temperature and incubated with 100 μl of anti-RBD monoclonal antibodies (Invitrogen, P06DHuRb, twofold diluted in 1% BSA in DPBS-T from 2.5 to 0.02 µg/ml concentration) to spike variants coated wells for 1 h at 37 °C. All wells were washed and incubated 100 μl of goat anti-rabbit IgG-HRP antibody at dilution of 1 in 5000 in blocking buffer. The level of anti-RBD bound to each spike variants were estimated by 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate. The level of binding of IgGs in each sera sample to spike variants were quantified by measuring the optical density (OD) at 450 nm in i3x plate reader (Molecular Devices, San Jose, CA). OD of each sera sample for spike variants was subtracted to respective spike variants blank OD (without sera). The percentage of maximum anti-RBD antibody binding was calculated by the following formula: [OD of concentrations/OD of highest concentration of spike variants)] × 100%. The Half maximal effective concentration (EC₅₀) of each variant was calculated by agonist versus normalized response–variable slope model.

Oxidized and total glutathione levels were assessed by using the GSH-Glo glutathione assay (V6611, Promega). HepG2 cells were plated at 10,000 cells per well in white 96-well plates. Cells were stimulated for 1, 6, 12, and 24 h before addition of glutathione detection reagents. Oxidized and total glutathione levels were assessed by the bioluminescent signal on the i3x plate reader (Molecular Devices) with an integration of 1,000 ms. Data is expressed as the ratio of oxidized glutathione over total glutathione. Statistics were carried out on Prism 9.1 (Graphpad).