Nadp nadph glotm assay

LN-308 cells were treated for 24 h with 2.5 nM Tunicamycin (or DMSO as a control) and subsequently washed with PBS prior to harvesting. For each assay, 4 × 10⁴ cells were resuspended in 50 µl of PBS and lysed with an equal volume of a solution containing 1% DTAB in 0.2 N NaOH. NADPH and NADP⁺ concentrations were determined with the NADP/NADPH-Glo^TM assay (Promega) according to the manufacturer’s instructions. In brief, samples were split into aliquots of 50 µl each and subjected to treated either with acid (25 μl of 0.4 N HCl to determine NADP⁺ levels) or base (to determine NADPH levels). Samples were incubated for 15 min at 60 °C, cooled to room temperature and neutralized with Tris. 50 µl of each sample were then mixed with an equal volume of NADP/NADPH-Glo^TM detection reagent and after incubation for 45 min subjected to luminescence measurement using a Centro XS3 luminometer (Berthold).

Purified neutrophils were cultured and treated with LPS (100 ng/ml) and/or TPM (70 ng/ml equi-nicotine units) overnight, followed with or without stimulation of PMA (20 ng/ml) for 15 min, then subjected to NADP/NADPH-Glo^TM assay, according to the manufacturer's instructions (Promega, Madison, WI).

NAD⁺/NADH-Glo^TM Assay (Promega, Madison, WI), NADP⁺/NADPH-Glo^TM Assay (Promega, Madison, WI), Reactive oxygen species (ROS)-Glo^TM (Promega, Madison, WI, USA), Glutathione (GSH)–Glo^TM Assay (Promega, Madison, WI, USA) were run based on same procedure. BT474, MCF7 ESR1^Y537S and T47D ESR1^Y537S cells were seeded at a concentration of 2 × 10³ cells/well in 96-well plates in corresponding phenol red-free media. Next day, cells were treated with endocrine agents alone or in combination with SEL in corresponding concentrations, and they were incubated with drugs for 24 h. All assays were run according to the supplier’s instructions for use of products. After 24 h incubation with drugs, luminescence values were measured using a Cytation5 plate reader (BioTek, Winooski, VT, USA). Standard curve and sample values were analyzed after subtraction of values from blank sample, and all statistical analyses were done by using Graphpad© Prism8 software (RRID:SCR_002798, GraphPad Software Inc., La Jolla, CA, www.graphpad.com). All experiment conditions had six technical repeats and experiments were repeated in six technical repeats at least for two times.