Anti gr 1 microbeads

Cannabidiol, SR141716A (SR1, CB1 antagonist) and SR144528 (SR2, CB2 antagonist) were provided by National Institute of Drug Abuse. The monoclonal antibodies (mAbs), FITC-conjugated anti-CD11b (clone: M1/70), anti-Ly6C (HK1.4), PE-conjugated anti-Gr-1 (anti-Ly6G/Ly6C, clone: RB6-8C5), anti-Ly6G (clone: IA8), anti-CD3, anti-CD4, anti-CD8, anti-CD31, anti-CD11c, anti-F/480, anti-Ki-67, Alexa 647-conjugated anti-CD11b and purified anti-CD16/CD32 (mouse Fc receptor block) were from Biolegend (San Diego, CA). The anti-arginase Ab was obtained from BD Transduction Laboratories. The anti-Gr-1 microbeads, magnetic sorting columns and equipment were from Miltenyi Biotech. Adenosine (A_2A) receptor antagonist 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), PPARγ antagonist 2,2-Bis[4-(2,3-epoxypropoxy)phenyl]propane (Bisphenol A diglycidyl ether or BADGE) and PPARγ agonist 5-[[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione (troglitazone) were purchased from Tocris Bioscience. Cell culture grade concanavalin A, L-arginine, L-ornithine standard, Ninhydrin reagent, red blood cell lysis buffer and all other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO).

MDSC were enriched by magnetic sorting using anti-Gr-1 micro beads according to manufacturer’s instructions (Miltenyi Biotech). Enriched cells were found to be >90% pure for CD11b and Gr-1 expression as determined by FACS analysis. For some experiments, CD11b⁺Gr-1⁺ MDSC, granulocytic and monocytic MDSC subtypes were purified by sorting (>90% purity) on FACS Aria cell sorter (BD Biosciences) after labeling with appropriate fluorescently conjugated mAbs.