Polarstar omega fluorescence plate reader

Cells (5 × 10⁶ cells per ml) were plated on a 96-well plate, and incubated with PMA or LPS for 2 h at 37 °C. The reactions were terminated with 4% (w/v) PFA (Sigma-Aldrich) overnight. Cells were permeabilized with 1% Triton X-100 for 25 min and then blocked with 2.5% (w/v) BSA in PBS for 1 h. 8-oxoG was probed for using mouse anti-8-Oxoguanineantibody (MAB3560, Millipore Sigma) at a 1:250 dilution. Plate was Imaging was done using Olympus IX81 inverted fluorescence microscope with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU ×1 spinning disk confocal scan head. Fluorescence was measured using POLARstar OMEGA fluorescence plate reader (BMG Labtech; excitation = 485 nm, emission = 525 nm).

SYTOX Green dye (5 μM; Thermo Fisher Scientific) was added to cells (5 × 10⁵ cells per ml). Cells were seeded on a 96-well plate. Inhibitors were added to the cells, followed by a 1 h incubation at 37 °C. Media control (negative control), NOX-dependent agonists and Triton X-100 (positive control) were then used as cell activators. The inhibitors used were APE inh 1 (CRT0044876, Sigma), APE inh 2 (APE1 Inhibitor III, EMD Millipore), PARP1 inh 1 (BSI201, Sigma), PARP inh 2 (PJ34, EMD Millipore), and LIG inh (L189, Tocris). They were dissolved in DMSO and diluted in media before being added to the samples at the required concentrations. Fluorescence of SYTOX Green-DNA interaction was measured using POLARstar OMEGA fluorescence plate reader (BMG Labtech; excitation = 485 nm, emission = 525 nm) every 30 min for 240 min Plotted Data represents NETosis levels. NETosis index was determined by dividing the fluorescence reading of each treatment by the reading of 1% (v/v) Triton X-100-treated cells.

SYTOX Green was added to cells (5 × 10⁵ cells per ml) at a concentration of 1:1000 (5 μM; ThermoFisher Scientific). The cells were seeded on a 96-well plate and then incubated with various inhibitors for 1 h at 37 °C. The cells were then activated with either media control (−ve control), different dosage of UV or known NOX-dependent (25 nM PMA) and -independent agonists (4 µM A23). The NETosis kinetics was assessed by measuring the fluorescence of SYTOX Green-DNA interactions using POLARstar OMEGA fluorescence plate reader (BMG Labtech; excitation = 485 nm, emission = 525 nm) every 30 min for 240 min. Maximal levels of DNA release were determined by lysing cells with 1% (v/v) Triton X-100. NETosis index was determined by dividing the SYTOX Green reading of each condition by the reading of 1% (v/v) Triton X-100-treated cells taken at 240 min.