Synergy neo2 multi mode assay microplate reader

Neutralization assays were performed using 293T-ACE2 cells (BEI NR52511) as previously described³⁸ (link) with few modifications.⁷⁰ (link) Briefly, rabbit sera were inactivated for 30 min at 56°C. Serial dilutions of the inactivated sera were incubated for 1 h at 37°C with SARS-CoV-2 PsV and subsequently added to 293T-ACE2 cells (BEI NR52511) seeded in Poly-L-lysine (Sigma-Aldrich) coated plates 24 h prior to the experiment. A final concentration of 5 μg/ml polybrene (Sigma-Aldrich) was added to the PsV-sera mixtures. After 48 h of incubation at 37°C, neutralization was monitored by adding 50 μl of Britelite plus reagent (PerkinElmer) to 50 μl of cells for 2 min. Supernatants were transferred to a 96-well white plate (Sigma-Aldrich) to measure luminescence in relative light units (RLUs) using a Synergy Neo2 Multi-Mode Assay Microplate Reader (Biotek Instruments). Absorbance data were converted to % inhibition using the same formula as used in the sVNT. Plotted data are the average of at least three replicate measurements. Data were fitted using 4PL regression constrained at top = 100% and bottom = 0% in GraphPad Prism 9.3.1 for determination of neutralization titers.

To measure the ATP hydrolysis activity of ATP synthase, enzyme-coupled ATPase activity assays were performed in 96-well plates with 160 μL buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% [v/v] glycerol, 5 mM MgCl₂, 0.2 mM NADH, 2 mM ATP, 1 mM phosphoenol pyruvate, 3.2 units pyruvate kinase, 8 units lactate dehydrogenase, 0.05% [w/v] DDM, DMSO [v/v] 2%, 2 nM ATP synthase). NADH concentration was monitored at 24 °C with a Synergy Neo2 Multi-Mode Assay Microplate Reader (BioTek) measuring absorbance at 340 nm. Inhibition with ammocidin and apoptolidin was determined by adding different concentrations of the inhibitors to assay mixture. Assays were performed in triplicates with two independently purified batches of yeast ATP synthase.