Apyrase grade 7

Aprotinin, nucleotides, apyrase grade VII, phenylmethanesulfonyl fluoride (PMSF), ticlopidine, and malachite green were purchased from Sigma-Aldrich (Oakville, ON, Canada). Tris was obtained from VWR (Montreal, QC, Canada). DMEM was obtained from Invitrogen (Burlington, ON, Canada). Fetal bovine serum (FBS) and antibiotics-antimycotics solution were from Wisent (St-Bruno, QC, Canada). Formalin and acetone were obtained from Fisher Scientific (Ottawa, ON, Canada). OCT freezing medium was purchased from Tissue-Tek, Sakura Finetk (Torrance, CA).

NETosis measurements were made as follows; 10⁵ BMDNs were seeded in 96-well assay plate (Corning #3603, Sigma Aldrich). 50 nM PMA or 1 μM A23187 (Sigma Aldrich, #P1585, #C7522 respectively) were added to wells in duplicates/triplicates with DMSO control included. The cell impermeable SYTOX green nucleic acid stain (5 µM, ThermoFisher Scientific #S7020) was added. SYTOX green stain measurement was performed using Spark Microplate Reader (Tecan) fitted with monochromatic filter 504/523 nm. 1 μM BB-FCF was used for Panx1 inhibition with appropriate controls. ATP scavenger apyrase grade VII (50 U/ml, Sigma Aldrich, #A6535), P2Y₂ receptor antagonist AR-C 118925XX (500 nM, Tocris, #4890) and CD39 ARL 67156 trisodium salt (100 μM, Tocris, #1283) were used for the purinergic signalling experiments involving ATP release. 10 mM N-acetyl cysteine (NAC, Sigma Aldrich #A0737) was used for ROS inhibition. For time-lapse imaging, SYTOX green dye (5 μM) was added to BMDNs seeded in 96-well assay plate. Cells were stimulated with 1 μM A23187 for 30 min in the presence of appropriate controls. Images were acquired at 10 minutes intervals for 5 h using the ImageXpress Micro Widefield High Content Screening System (Molecular Devices, San Jose, USA). MetaXpress 2.0 software was used to reconstitute images for video analysis.

Isolated CD4 T cells or human PBMC cells were suspended at a density of 1 × 10⁶ cells/ml in RPMI1640 + 10 mM HEPES + 0.1% BSA. After 1 h of serum-starvation, 50 μl of the cell suspension were added to 5 μm pore size 96 well inserts (Corning, Acton, MA, USA). The lower wells contained either 250 μl medium alone (RPMI1640 with 10 mM HEPES and 0.1% BSA) or medium plus CCL19, CCL21, CCL4, SDF-1α, CXCL9, CXCL10, and CCL5 (all 100 ng/ml, Peprotech). In some cases, apyrase grade VII (20 units/ml, Sigma-Aldrich) or carbenoxolon (5 μM, 30 min pretreatment, Cayman Chemicals) was added together with agonists. Cells were allowed to transmigrate for 3 h at 37 °C and 5% CO₂, then transwell plates were kept on ice for 20 min and centrifuged at 180 × g for 3 min. Inserts were discarded, and the number of cells transmigrated compared to input was determined by flow cytometry. “% input” was calculated as (number of transmigrated CD4 T cells/number of CD4 T cells added to the upper well) × 100.

Compound 1 was prepared as previously described [18] (link). Adenosine 5′-(P¹-thio-P²,P³-chloromethylenetriphosphate), compound 2 was isolated as a minor byproduct during the synthesis of 1 and was characterized by ¹H, and ³¹P NMR and liquid chromatography-mass spectrometry (LCMS). Adenosine 5′-thiomonophosphate (thioAMP), MRS2179, probenecid, adenosine 5′-(β,γ-methylenetriphosphate) (β,γ-CH₂-ATP), ethylene glycol tetraacetic acid (EGTA) and apyrase (grade VII) were purchased from Sigma-Aldrich (St. Louis, MO). D-Phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) was purchased from Calbiochem (La Jolla, CA). FLUO-4 was from Invitrogen (Carlsbad, CA), ADP from Bio/Data (Horsham, PA), CD41-phycoerythrin (PE)-Cy5 from Beckman Coulter (Fullerton, CA). The VASP phosphorylation kit was purchased from BioCytex (Marseilles, France).