Luminescence atp detection assay kit

The level of ATP was measured in freshly isolated PMNs using Luminescence ATP detection Assay Kit (Abcam) following the manufacturer’s instructions. Isolated resting WT and Nrf2 KO PMNs were seeded at 1 × 10⁵/well in a white flat bottom 96-well plate (Costar) without any stimulation. Samples were tested in triplicate. Luminescence was quantified using the multimode microplate reader (TristarTM LB941 Berthold) and then converted to ATP concentration (in μM) using standard curve.

To determine ATP consumption in in vitro SUMO and FAT10 activation assays, the Luminescence ATP Detection Assay Kit (Abcam, ab113849) was used, as described by the manufacturer. Briefly, in vitro SUMO or FAT10 activation assays were performed with 0.3 nM AOS1/UBA2 and 3.3 nM SUMO-1 or 3.3 nM FAT10 or FAT10-AV in 1x in vitro buffer (20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 10 mM MgCl2, 4 µM ATP, and 0.1 mM DTT) for 30 min at 30 °C. Samples were subsequently treated as described by the manufacturer and luminescence was measured in a multiwell plate reader SPARK 10 M (TECAN). To be in a linear range, the minimal amount of ATP needed for in vitro SUMO activation had been determined to be 4 µM in a preceding titration experiment.