Gsh gssg glo luminescence based kit

Intracellular NADP⁺/NADPH ratios were measured using NADP⁺/NADPH-Glo™ kit (#G9081, Promega) according to the manufacturer. DRGs were seeded in 96-well plates. Following treatment, DRGs were lysed in NADP⁺ or NADPH lysis buffer (60°C/15 min) and spun at 13,000 g. Supernatants were loaded into white wall 96-well plate, mixed with detection reagent and incubated in the dark (22°C/30 min). NADP⁺ and NADPH concentrations were derived from standard curves (Magellan™ software, TECAN, San Jose, CA). Measurements of total and reduced glutathione levels were carried out using the GSH/GSSG-Glo™ luminescence based kit (V6611, Promega™) following manufacturer’s guide using 96-well white wall-plate format. Three sets of independent experiments were carried out and luminescence was read on TECAN FL200 plate reader.

Assessment of reduced intracellular glutathione (GSH) levels was with Thioltracker Violet (Life Technologies) cell-permeant thiol-reactive fluorescent probe as described [40 , 41 (link)]. DRG neurons were seeded in black wall 96-well plates (10⁴/well). After treatments cultures were washed with PBS and freshly prepared 10 μM Thioltracker Violet in DPBS was added for 30 min/37°C in the dark. Following 2 washes with PBS, residual fluorescence was measured using the FL200 Tecan (excitation at 415 nm/emission 530 nm). Measurements of total and reduced glutathione levels were carried out using the GSH/GSSG-Glo luminescence based kit (V6611, Promega^™) following the manufacturer’s guide using 96-well white wall-plate format and TECAN FL200 plate reader.