Anti mouse cd80 pe

Flow cytometry analysis was performed using the following antibodies (BD Bioscience): Anti-mouse CD3-PE/Cy7, Anti-mouse CD11c-PE/Cy7, Anti-mouse CD8-PE/Cy5, Anti-mouse CD44-PE, Anti-mouse CD62L-FITC, Anti-mouse PD-L1-PE, Anti-mouse CD326 PE/Dazzle, Anti-mouse CD4-PE/Dazzle, Anti-mouse CD80-PE, Anti-mouse CD86-PE/Dazzle, and Anti-mouse CD45 FITC. All samples were suspended in FACS buffer and stained with 1 µL antibodies for 30 min at 4°C in darks, then washed twice and resuspended in FACS buffer before analysis. For γ-IFN detection, Human Cytometric Bead Array (CBA) kit and mouse CBA kit (BD Bioscience, USA) were used. The antibody dilution concentration was 1:100. Samples were analyzed with BD Accuri C6 (BD Bioscience, USA) and CytoFLEX (Beckman, USA).

Anti-PD-L1 (ab213480, Abcam), anti-GAPDH antibody (2118, CST), anti-Phospho-TBK1/NAK antibody (5483, CST), HRP-conjugated secondary antibodies (7074, CST), anti-PD-L1(BE0101, BioXcell), anti-mouse CD16/32 antibody (553,141, BD Pharmingen), Fixable Viability Stain (565,388, BD Pharmingen), anti-CD80-BV421(562,611, BD Pharmingen), anti-CD86-PE-Cy7(560,582, BD Pharmingen), anti-mouse PD-1-PE (135,206, BioLegend), anti-mouse LAG3-PE-Cy7 (125,226, BioLegend), anti-mouse TIM3-BV421 (119,723, BioLegend), anti-mouse FOXP3-BV421 (126,419, BioLegend), anti-mouse Ki67-PE-CY7 (652,426, BioLegend), anti-mouse PD-L1-PE (124,308, BioLegend), anti-mouse CD11b-FITC (101,206, BioLegend), anti-mouse Ly6G-PE (551,461, BD Pharmingen), anti-mouse Ly6C-PE-Cy7 (128,018, BioLegend), anti-mouse F4/80-BV421 (123,132, BioLegend), anti-mouse CD206-APC (141,708, BioLegend), anti-mouse TNFα-PE (554,419, BD Pharmingen), anti-mouse CD3-BV605 (100,237, BioLegend), anti-mouse CD11C-APC (117,310, BioLegend), anti-mouse MHC-II-BV421 (107,632, BioLegend), anti-mouse NK1.1-PE-Cy7 (552,878, BD Pharmingen), anti-mouse CD80-PE (552,769, BD Pharmingen).

Bone marrow-derived macrophages were transduced with a control vector (pMX-FLAG-IRES-EGFP) or the constitutively active form of STAT5A (pMX-FLAG-IRES-EGFP-STAT5A1*6), differentiated to osteoclasts in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL), and collected. The collected cells were stained with PE-anti-mouse CD80 (BD Pharmingen^TM, San Diego, CA, USA), PE-anti-mouse CD 86 (BD Pharmingen^TM), PE-anti-mouse MHC class II (eBioscience, San Diego, CA, USA), and APC-anti-mouse CD11c (eBioscience) for 20 min at 4 °C. Stained cells were analyzed using a Navios flow cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA).