P1132

Bovine PMN suspended in RPMI 1640 medium were confronted with B. besnoiti tachyzoites (4 h, 37°C, 5% CO₂) at a PMN/tachyzoite ratio of 1:6 (2×10⁵ PMN:1.2×10⁶ tachyzoites, 96-well format) in the presence of non-modified ATP (P1132, Promega, USA), non-hydrolyzable ATP (ATPγS; 0.05-50 µM; 4080, Tocris, UK), or purinergic receptor antagonists (see Table 1) at a concentration range of 0.1-100 µM. After incubation, sample supernatants were analyzed for “cell-free” NETs. The remaining cells at the well bottoms were estimated for “anchored” NETs according to (44 (link)). Therefore, picogreen (Invitrogen, Eugene, USA, 1:200 dilution in 10 mM Tris base buffered with 1 mM EDTA, 50 μl/well) was added to each supernatant or pellet sample. Extracellular DNA was quantified by picogreen-derived fluorescence intensities using an automated microplate reader (Varioskan, Thermo Scientific) at 484 nm excitation/520 nm emission as described elsewhere (26 (link), 30 (link)).

The intracellular ATP levels of control and treatment (arsenate) groups were measured using a BacTiter-Glo™ Microbial Cell Viability Assay kit (Catalog# G8230, Promega Corporation, Madison, WI) according to manufacturer’s guideline. Fresh LB media was used to measure the background luminescence. Standard curves were generated by measuring the luminescence of ATP solutions at known concentrations. These solutions were prepared by serially diluting rATP (Promega catalog# P1132) in fresh LB.

ISC-enriched organoids in 3D Matrigel culture were passaged to a 48- or 96-well plate and cultured with ENR or ENR+CD media containing DMSO or each drug for 6 d. DMSO- or drug-containing media were changed every other day. On day 6, cells were washed with basal media twice and treated with basal media with or without 10 μM carbachol for 3 h in a CO₂ incubator at 37 °C. Conditioned media was collected and used for lysozyme assay (Thermo Fisher, E-22013) following the manufacturer’s instruction. The fluorescence was measured using excitation/emission of 485/530 nm. CTG 3D Reagent was added afterward, and the cell culture plate was incubated on an orbital shaker at RT for 30 min to induce cell lysis and to stabilize the luminescent signal. The solution was replaced to a 96-well white microplate, and luminescent signals were measured by a microplate reader (infinite M200, Tecan). The standard curve was prepared by diluting recombinant ATP (Promega, P1132). For both assays, a polynomial cubic curve was fitted to a set of standard data, and each sample value was calculated on the Microsoft Excel.