Nadph glo assay kit

Cells were lysed in 0.1 M NaOH with 0.5% DTAC for NADPH determination at 0.1 × 10⁶ cells per well. Relative NADPH levels were then determined using the NADPH-Glo assay kit (Promega) as per manufacturer’s instructions.

Magnetically sorted neutrophils were seeded at 0.25–0.3 × 10⁶ cells per well of a 96-well luminescence plate in complete seahorse media (glucose 25 mM and L-glutamine 2 mM) and incubated for 30 min at 37 °C. For H₂O₂ measurements 400 µM luminol was added to the media and cells were stimulated for 20 min with 2DG (100 mM), Rotenone (100 nM) and antimycin A (1 µM) or etomoxir (100 µM) following which cells were stimulated with PMA (1 µg/ml) and immediately measured for luminescence in a 96-well luminescence plate reader and luminescence readings were measured every 6 min for 6 h. For ATP and NADPH measurements, cells were lysed in passive lysis buffer (Promega) or 0.1 M NaOH with 0.5% DTAC for ATP or NADPH determination respectively. Relative ATP was determined using the ATP assay kit (Abcam/Thermo-Fisher) and NADPH was determined using the NADPH-Glo assay kit (Promega) as per manufacturer’s instructions.

Magnetically sorted pTRMØ were seeded at 0.2–0.4 × 10⁶ cells per well of a 96-well luminescence plate in complete media and incubated for 2 h at 37 °C. The media was replaced with minimal media (DMEM with no pyruvate, no phenol red, no glucose, no glutamine, 0.2 units/ml penicillin, 100 µg/ml streptomycin), or Seahorse media (no glutamax, no glucose, no glutamine). Neutrophils were then added to separate wells on the plate and cells were supplemented with ±glucose (25 mM) or ±l-glutamine (2 mM), or additional treatments as indicated (including 400 µM luminol). The plate was incubated at 37 °C for 30 min, after which ±zymosan (50 μg/ml) was added. For luminol measurements, the plate was placed in a 96-well luminescence plate reader and luminescence readings were measured every 6 min for 6 h. Otherwise, cells were lysed in passive lysis buffer (Promega) or 0.1 M NaOH with 0.5% DTAC for ATP or NADPH determination, respectively. Relative ATP was determined using the ATP assay kit (Abcam/Thermo-Fisher) and NADPH/NADP ratios determined using the NADPH-Glo assay kit (Promega) as per manufacturer’s instructions.