Apc cy7 anti mouse ly 6g ly 6c gr 1

Carprofen (Rimadyl^® Injectable, 50 mg/mL) was purchased from Patterson Companies (Saint Paul, MN, USA). Pacific Blue™ anti-mouse CD3, FITC anti-mouse CD4, PerCP anti-mouse CD8a, PE anti-mouse/human CD11b, APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1), PE/Cy7 anti-mouse CD62L, APC anti-mouse/human CD44, APC anti-mouse CD25, Biolegend PE anti-mouse/rat/human FOXP3, PerCP anti-mouse CD11c, PE/Cy7 anti-mouse CD86, FITC anti-mouse I-A/I-E, and APC anti-mouse CD40 were purchased from Biolegend (San Diego, CA, USA). Mouse tumor dissociation kit (No. 130-096-730) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

For immunophenotyping macrophages, control and anti-CD47 mAb-treated mice were killed, and subcutaneous tumors were dissociated to single cells and stained with APC/Cy7 anti-mouse LY-6G/Ly-6C (Gr-1) (BioLegend), PE/Cy7 anti-mouse CD45 (BD Bioscience), PerCP/Cy5.5 anti-mouse/human Cd11b (Biolegend), APC anti-mouse F4/80 (eBioscience), Brilliant Violet 785™ anti-mouse I-A/I-E (MHC-II) (BioLegend), Brilliant Violet 605™ anti-mouse CD86 (BioLegend), PE anti-mouse CD206 (BioLegend), PE/Cy7 anti-mouse CD124 (IL-4R alpha) (BioLegend), and Alexa Fluor® 488 anti-mouse CD80 (BioLegend), all according to the manufacturers’ specifications. Endogenous mouse FcγII and FcγII receptors were blocked using 3% BSA containing Mouse BD Fc Block^TM (1 μg). Flow cytometric analysis was performed on a BD FACS ARIA II (BD) flow cytometer. Data analysis was performed using FlowJo Version 9.6.4 (Tree Star, Ashland, OR, USA). Live singlets were gated using FSC-W/FSC-H. Gates were drawn using Fluorescent Minus One control tubes.