Nad nadh glo assay

PBMCs (50.000 cells) purified from HD, CA, COV and COV/CA patients were cultured in completed RPMI medium (10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml Penicillin and 100 µg/ml Streptomycin), supplemented with 800 µM Nicotinamide (Duchefa Biochemie) or 100 µM Apigenin (Merk) or 500 µM Leucine (Merk) or cell culture media for 1 h. Intracellular NAD+ levels were then quantified via NAD/NADH-Glo Assays (Promega). Luminescence was quantified with microplate reader Synergy 2 (Bio-Tek instrument INC).

Schwann cells were directly plated on PLL coated 24 well plates and maintained in DMEM/5% horse serum (HS). Schwann and HEK 293T cells were treated with 100 μM vacor, 250 μM 3-AP, or vehicle controls, and collected in media at 0 h and 72 h after treatment. Protein was extracted using Pierce™ IP Lysis Buffer supplemented with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, and a Bicinchoninic acid (BCA) assay carried out to determine protein concentration. All samples were diluted to 0.5 μg μL⁻¹. The NAD-glo assay was performed according to manufacturer’s instruction of the NAD⁺/NADH-Glo™ assay by Promega (G9071). In short, 25 μL of sample were incubated with 12.5 μL 0.4 M HCl at 60°C for 15 min, before 12.5 μL 0.5 M Tris base were added. 10 μL NAD⁺ standards or sample were then mixed with 10 μL NAD-glo master mix (1 mL luciferin detection reagent, 5 μL reductase, 5 μL reductase substrate, 5 μL NAD⁺ cycling enzyme, 25 μL NAD⁺ cycling substrate) and incubated at RT for 40 min. Luminescence was read using a GloMax^® Explorer microplate reader and NAD⁺ concentrations determined relative to a NAD⁺ standard curve.

Intracellular ATP and ADP were extracted as previously described [80 (link)]. ATP/ADP ratios were measured using a luciferin-luciferase ATP kit (Microbial ATP Kit HS, BioThema AB, Sweden). Intracellular NADH and NAD⁺ was extracted as previously described [81 (link)]. The NADH/NAD⁺ ratio was quantified by a luciferase assay provided by the kit NAD⁺/NADH-Glo Assay (Promega). The luminescence from each assay was measured using the Infinite® M1000 PRO microplate reader (TECAN) with the 96-well microplates from Greiner Bio-one (Cat. No. 655901).

Separate measurements of oxidized (NAD⁺⁾ and reduced (NADH) forms were assessed with the NAD/NADH-Glo™ Assay (Promega) following the manufacturer's protocol. Briefly, K-562 cells were seeded at 3 × 10⁵ cells/mL and treated with the indicated concentration of MAC681 for the indicated time. After the treatment, the concentration of cells was quantified by trypan blue exclusion assay, and cells were centrifuged and resuspended in 1 × PBS at a concentration of 5 × 10⁵/mL. The cells were lysed in the base solution with 1% dodecyltrimethylammonium bromide, then split into separate wells for acid (NAD⁺) and base (NADH) treatments. After 15 min heating at 60°C, the samples were incubated for 10 min at room temperature. 25 μl of Trizma® base (Sigma-Aldrich) was added to each well of acid-treated samples, and 50 μl of HCl/Trizma® solution was added to each well of base-treated samples. 50 μl of NAD/NADH-Glo™ Detection Reagent was added to each well. Luminescence was recorded after 60 min of incubation at room temperature, and NAD⁺ to NADH ratio was calculated (SpectraMax ID3 Multi-Mode Microplate Reader, Molecular Devices, LLC, San Jose, CA, USA).

Cells were plated in a 96-well plate and treated with the indicated concentration of phenformin for 24 h. Using NAD⁺/NADH Glo Assay (Promega), the NAD⁺/NADH ratio was calculated [6 (link)].