Atp disodium salt hydrate

Filaments (or, depending on ligand state, free octamers) were prepared by diluting aliquots of purified hIMPDH2 in activity buffer (20 mM HEPES, 100 mM KCl, 1 mM DTT, pH 7.5) to 2 μM in the presence of varying concentrations of ATP, GTP, IMP, and/or NAD⁺ and incubating for 30 min at 20°C. Nucleotide stocks were prepared using ATP disodium salt hydrate (Sigma A2383), GTP sodium salt hydrate (Sigma G8877), IMP disodium salt hydrate (Acros AC226260050), and β-Nicotinamide adenine dinucleotide hydrate (Sigma N6522), For all buffers, protein preparations, and nucleotide stocks, final pH was checked and titrated if necessary to 7.5 with KOH or HCl.

Monocytes from individuals heterozygous for rs257174 were purified from PBMCs by positive selection using MACS human CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions. Monocytes were plated at 0.5 × 10⁶ cells/well in 24-well tissue culture plates and then incubated with or without cell culture-grade ATP disodium salt hydrate (Sigma-Aldrich) in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml of penicillin and 100 μg/ml of streptomycin. For the inhibition of ATP the incubation was carried out in the presence of 10 μM of the purinergic receptor inhibitor A-438079 hydrochloride (Tocris Bioscience). Monocytes were incubated for 2 h before harvesting using cell scrapers. The treated monocytes were stored in TRIzol (Invitrogen Life Technologies) at -80°C until further analysis.

P2X₇R inhibitors AZ10606120 dihydrochloride and AZ11645373, A438079 hydrochloride (Tocris Bioscience) were dissolved in DMSO and used at concentrations noted in the results. BzATP triethylammonium salt (Tocris Bioscience) was dissolved in sterile water and used at millimolar concentrations. Nigericin (Calbiochem) was used at 10 μM concentration for all in vitro assays. Apyrase (Sigma Aldrich) was used at 10 U ml⁻¹. ATP disodium salt hydrate (Sigma Aldrich) was dissolved in water and used at micromolar concentrations. Ultrapure LPS (Adipogen) was dissolved in endotoxin free water and used at 500 ng ml⁻¹ for in vitro assays.

Overnight cultures were diluted 1/40 into 40 ml of M9 medium with carbon source and that lacked casamino acids. Bacteria were shaken at 250 RPM and at 37°C for 2 hours in a 50-ml conical tube. Cultures were then concentrated 2× in fresh diluted (3:1) M9 medium that contained the carbon source and casamino acids. One hundred microliters of these cultures was added to the wells of an opaque walled 96-well plate, overlaid with two Breath-Easy filters (Sigma-Aldrich, St. Louis, MO, to prevent evaporation), and was shaken at 250 RPM/37°C for 1 hour whereupon they reached OD₆₀₀ = ~0.075. [ATP] was measured using a BacTiter-Glo assay (Promega, Madison, WI) according to the manufacturer’s recommendations. ATP (quantified using luminescence) and OD₆₀₀ were measured in a microplate reader. Using a lower initial density of bacteria (1/400) to measure [ATP] does not affect the concentration of ATP measured (fig. S2—note that these bacteria were concentrated 10× before the measurement of ATP to account for differences in density). A standard curve prepared using pure ATP (ATP disodium salt hydrate, Sigma-Aldrich) was used to quantify the ATP concentration. Additional details on measuring ATP during heat shock, in the presence of protease inhibitor and in stationary phase, can be found in SM Methods.