Nad nadh glo kit

4T1 cells were seeded per well in 12-well plates for 24 h. After various treatments, the cellular NADH concentrations was determined using the NAD/NADH-Glo™ kit (Promega) by chemluminescence using a microplate reader. Each group was determined as duplicates of quadruplicate.

Cells were plated in 96-well plates (15,000 cells/well). Twenty-four hours later, the media were supplemented with ZZW-115, olaparib and or 5-FU. NAD⁺ and NADH levels were determined by using Promega NAD⁺/NADH-Glo kit (G9071) using manufacturer’s instructions. After treatment, media was exchanged to PBS and cells were lysed in 1 % DTAB (Dodecyltrimethylammonium bromide) and 0.2 M NaOH. Cell lysate was immediately divided into two 50 μl aliquots. One aliquot, to measure NADH was left unmodified, and the other aliquot, to measure NAD⁺, was supplemented with 25 μl of 0.4 M HCl. Both aliquots were heated at 60 °C for 20 min to destroy reduced or oxidized nucleotides and then, incubated 10 min at room temperature. The first one was complemented with 50 μl of HCl/Trizma solution and the second one with 25 μl of Trizma base. Finally, NAD/NADH-Glo™ Detection Reagent was added to the samples and they were incubated at room temperature for 45 min. NAD⁺ and NADH levels were determined using a Tristar multimode microplate reader (Berthold).

50,000 cells were seeded per well in 6-well plates in 2 mL full DMEM. After 24 h, cells were rinsed in PBS, trypsinized, and counted. 5000 cells per condition were assayed per well in white-walled 96-well plates using the Promega NAD/NADH-Glo kit. Cells of independent experiments were seeded and assayed on separate days.