NAD+ measurements

The level of NAD⁺ and NADH in cells was measured using the Enzychrome NAD⁺/NADH colorimetric assay kit (BioAssay Systems) as we have described previously^16,103. Briefly, cells were seeded in a 6 well plate at the density 2 × 10⁵ cells per well for NAD⁺ measurements and 3 × 10⁵ cells per well for NAD⁺ pool measurements (NAD⁺ and NADH). 24 hrs later, cells were treated with the indicated NAD⁺ precursors (100 μM) in the presence or absence of FK866 (30 nM). Following 24 hrs of treatment, cells were harvested and a suspension of 2 × 10⁵ cells was divided in half for measuring NAD⁺ and NADH respectively or a suspension of 1 × 10⁵ cells was used for the NAD⁺ measurement only. Cell pellets were homogenized using plastic pestles and the extraction of NAD⁺ and NADH was performed in the provided lysis buffers. Extracts were heated at 60 °C for 5 min and neutralized with the provided buffers. Samples were spun down and the supernatant was immediately used for measurements of NAD⁺/NADH content using a Molecular Devices VersaMax™ tuneable plate reader at 565 nm.