5 bdbd

Mice were immunized i.p. with 50 μg NP₁₈-KLH in alum. To induce Cre activity, adults were fed tamoxifen-laced cow (ENVIGO) for 4 weeks before initiating experiments. Mice were treated with 5-BDBD (4.25 mg/kg i.v.; R&D), Suramin (2.0 mg/kg i.v.; Abcam), or PPADS (2.5 mg/kg i.v.; Santacruz)^{45 (link)} in a volume of 30μl. 5-BDBD and the P2rX7 inhibitor A438079 (R&D Systems) were each used in vitro at a final concentration of 50μM. All P2rX inhibitors were dissolved initially in DMSO.

Fluo-4 AM, Rhod-2 AM, MitoTracker Red CM-H2Xros, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Molecular Probes (Thermo Fisher Scientific). Recombinant human SDF-1α, NF340 (P2Y11 antagonist), NF546 (P2Y11 agonist), 5-BDBD (P2X4 antagonist), CSC (A2a antagonist), MRS1754 (A2b antagonist), and H89 (PKA antagonist) were from R&D Systems, and cAMP-AM was from BIOLOG Life Science Institute. In all experiments, 5-BDBD, NF340, and NF546 were used at concentrations (1-10 μM) at which they were reported to be specific for the respective receptor (36 (link), 64 (link), 65 (link)). Anti-human CD4-allophycocyanin (clone: OKT4) and anti-human CD69 FITC (clone: FN50) antibodies were purchased from Biolegend. Mouse anti-human CD3 (clone: HIT3a) antibodies were from BD Pharmingen. All other reagents were purchased from Sigma-Aldrich unless stated otherwise.

Carboxyfluorescein succinimidyl ester (CFSE), Fluo-4 AM, MitoTracker Red CM-H2Xros, MitoTracker Green FM, and Tubulin Tracker Deep Red were purchased from Thermo Fisher Scientific (Waltham, MA). The P2Y11 receptor antagonist NF340, the P2Y11 receptor agonist NF546, the P2X4 receptor antagonist 5-BDBD, the PKA inhibitor H89, and nocodazole were from R&D Systems (Minneapolis, MN), while cell permeable cAMP-AM was from BIOLOG Life Science Institute (Bremen, Germany). Anti-human CD4-allophycocyanin (clone: OKT4), anti-human CD69 FITC (clone: FN50), and anti-human CD11b Alexa Fluor 488 (clone: ICRF44) antibodies were purchased from Biolegend (San Diego, CA). Mouse anti-human CD3 (clone: HIT3a) and anti-human CD28 (clone; CD28.2) antibodies were from BD Pharmingen (Franklin Lakes, NJ) and goat anti-mouse IgG Fc antibodies were from Pierce (Thermo Fisher Scientific). Latrunculin B and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.