Pyruvate assay kit

Pyruvate was measured using either an enzymatic assay (most samples) or an HPLC-based assay (medium from 0.2% oxygen experiments). For the enzymatic assay, medium samples were diluted 20-fold in PBS. Pyruvate concentration was determined using the Pyruvate Assay Kit (Cayman). Medium samples did not require deproteinization, otherwise the samples were analyzed according to the manufacturer’s protocol. Standards were prepared in PBS. For the HPLC assay, 2-oxovaleric acid was added to medium samples as an internal standard. Samples were subsequently deproteinized with 2 volumes of ice-cold acetone. Supernatants were evaporated to <50% of the starting volume at 43 °C in a SpeedVac concentrator (Thermo Savant) and reconstituted to the starting volume with HPLC-grade water prior to derivatization. Samples were derivatized 1:1 by volume with o-phenylenediamine (25 mM in 2 M HCl) for 30 min at 80 °C. Derivatized pyruvate was separated with a Poroshell HPH C-18 column (2.1×100 mm, 2.7 μm) on an Infinity II high-performance liquid chromatography system with fluorescence detection of OPD-derivatized α-keto acids as described previously (Guarino et al., 2019 (link)).

Pyruvate levels were measured using the Pyruvate Assay Kit (Cayman) following manufacturer’s recommendation. Briefly, the cells were washed with PBS and then centrifuged at 10,000×g for 5 min. The supernatant was discarded, 0.5 ml of 0.25 M metaphosphoric acid (MPA) added to the cell pellet and placed on the ice for 5 min to deproteinate the sample. The suspension was centrifuged at 10,000×g for 5 min, the supernatant removed and 25 μL of potassium carbonate added to neutralize the acid. After centrifugation at 10,000×g for 5 min to remove any precipitated salts, supernatant was discarded. The sample was diluted in 1:2 with assay buffer that was subjected to microplate reader analysis at 590 nm. ATP levels were determined using ATP Detection Assay Kit (Beyotime Biotechnology) according to manufacturer’s instructions. Enough standard ATP solution was prepared and added to each well to exhaust the background. Subsequently, 20 μL of sample or standard was added to each well and a luminometer was used to measure the relative light unit (RLU) value. The ATP levels of samples were calculated by referring to the standard curve under the same conditions

Neutrophils (0.2 × 10⁶) were activated with Zy/op (100 µg/mL) for 6 h, at 37 °C and 5% CO₂. Lactate concentration in cultured supernatant was measured with the Enzymatic Lactate Kit (Labtest, 138).
For Dihydroxyacetone Phosphate, Glycerol-3-Phosphate, Triose Phosphate Isomerase, NADP/NDPH, glutathione (GSH/GSSG/Total), Pyruvate Kinase activity and Pyruvate analysis, we used commercial kits. Briefly, murine or human neutrophils (1 × 10⁶) were activated with Zy/op (100 µg/mL) for 1 h, at 37 °C and 5% CO₂. For human samples, neutrophils were pre-treated for 1 h with oxalate (3 mM) before being activated with Zy/op. Samples were centrifuged and prepared according to manufacturer’s instructions. The following kits were used: PicoProbe™ Dihydroxyacetone Phosphate (DHAP) Fluorometric Assay Kit, Glycerol-3-Phosphate (G3P) Colorimetric Assay Kit, Triose Phosphate Isomerase (TPI) Activity Colorimetric Assay Kit, NAD/NADH Quantitation Colorimetric Kit, Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit and Pyruvate Kinase Activity Colorimetric/Fluorometric Assay kit (Biovision, K673, K641, K670, K337 and K709, respectively); and Pyruvate Assay kit (Cayman, 700470).