Cd39 inhibitor pom1

Either MSCs or FACS-sorted conditioned monocytes were washed three times with MES buffer [0.025 M MES (Sigma) in 0.9% NaCl (Braun) pH 6.0] and cultured at 50,000 cells/well in the presence or absence of 5′AMP (Sigma) and the CD39 inhibitor POM1 (Tocris) or CD73 inhibitor APCP (ADP analog; Sigma) when indicated. After 2 h at 37°C, cells were centrifuged and supernatants harvested to freshly quantify Pi concentration using the malachite green phosphate colorimetric assay kit (BioVision), following the manufacturer’s instructions. 5′AMP and APCP alone were confirmed to yield negative values, and Pi production was calculated as the subtraction [Pi]_{cells+5′AMP} − [Pi]_{cells alone}. Alternatively, the 2 h-supernatant was snap frozen at −80°C and analyzed for Ado concentration using the Adenosine assay kit (BioVision), following the manufacturer’s instructions.

LPS (Escherichia coli serotype O55:B5) and adenosine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterated adenosine was from CDN Isotopes (Quebec, Canada). adenosine 5′-triphosphate disodium salt (ATP) was from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human M-CSF, IFNγ, and IL-10 were obtained from Peprotech (Rocky Hill, NJ, USA). Recombinant human GM-CSF and IL-4 were from Novartis AG (Basel, Switzerland). The RPMI 1640 medium, l-glutamine, streptomycin, penicillin, and heat-inactivated fetal calf serum (FCS) were obtained from Gibco, Thermo Fisher Scientific. CD39 inhibitor POM-1 was from Tocris Bioscience (Bristol, UK). The cell proliferation dye CFSE and calcium sensor Fluo-4, AM was from Molecular Probes, Thermo Fisher Scientific. Brilliant Violet 421-conjugated streptavidin used as a second step in flow cytometry analyses was purchased from BioLegend (San Diego, CA, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin calcium salt (ionomycin) from S. conglobatus and monensin A sodium salt (monensin) were purchased from Sigma-Aldrich.