Synergy h1 multi mode plate reader

For the lysosensor assay, cells were plated and left to attach overnight in a clear bottom 96-well black border plate (Corning). Next, cells were pulsed with media containing 0.2 mg/ml Lysosensor Yellow/Blue DND-160 dextran (LS) for 4 h, washed three times and chased in fresh media for 4 h. Any treatments as indicated were performed during chase period. For each experiment, a pH calibration curve was generated. To this end, cells were incubated for 10 min at 37 °C in sodium acetate-acetic acid calibration buffers (ranging from pH 4.2 to pH 5.2), and lysosomal pH clamped to the buffer pH by addition of 10 µM nigericin and 10 µM monensin. Dual-emission ratiometric measurements were performed with a Multi-Mode Plate Reader Synergy H1 (BioTek): excitation 329 nm/emission 440 nm and excitation 380 nm/emission 540 nm. Based on the emission intensity ratio 440 nm/540 nm, lysosomal pH values were calculated using the calibration curve.

Stock solutions of assay dye (monochlorobimane (MCB), 22 mM) and glutathione-S-transferase (50 units/mL) were prepared in PBS and stored protected from light at − 20 °C. The working solution was prepared using 12.8 μL of stock MCB and 80 μL of stock glutathione-S-transferase in 4 mL PBS and stored on ice. Samples were prepared as follows: cells were lifted mechanically using cell-lifters, washed twice and re-suspended in ice-cold PBS, mixed by vortex and incubated on ice for 30 min. Following two freeze thaw cycles using solid CO₂, the samples were sonicated 1 min on wet ice (S220 Ultra-sonicator from Covaris) and spun at 3000×g, 4 °C, for 5 min. Total protein concentration of supernatants was determined using Bradford assay. Samples and glutathione (GSH) standards (0–13 μM) were plated in 25 μL aliquots in a 96-well plate with clear bottom and black sides. Twenty-five μL of working solution was added to all experimental wells and protected from light for 15 min at room temperature. Fluorescence (ex 380 nm, em 461 nm) was measured using a Synergy H1Multi-Mode Plate Reader (Bio Tek). The amount of GSH detected in each sample was calculated using the regression curve obtained from the glutathione standards.