Anti mouse f4 80 pe cyanine 7

The harvested tumors of the mice were cut into pieces in DMEM medium containing 2% FBS on ice and then treated with 5 volume equivalent trypsin at 37℃ for 60 min. The samples were filtered and centrifuged at 600 g for 5 min. The cells were washed with PBS containing 2% FBS and co-incubated with sterile ZombieNIR dye and Fc receptor blocker at 4℃ for 15 min. Next, the cells were stained with the primary antibodies in dark for 30 min, including anti-mouse CD86-PE antibody (B334834, Biolegend), anti-mouse CD206-APC (B354282, Biolegend), anti-mouse F4/80-PE/Cyanine-7 (B342137, Biolegend), anti-mouse CD45-Brilliant Violet 421 (B343559, Biolegend), anti-mouse CD3-PE (B341466, Biolegend), anti-mouse CD4-FITC (B269033, Biolegend), anti-mouse CD8a-APC (B348048, Biolegend), anti-mouse CD11c-APC (B339313, Biolegend), anti-mouse I-A/I-E (MHC II)-PE/Cyanine-7 (B337001, Biolegend) and anti-mouse CD11b-FITC (B349919, Biolegend). After rinsing excess antibodies, the cells were immediately assessed by a flow cytometer (Beckman Coulter, USA).

For blood and tumor samples from mice, the following antibodies were purchased from BioLegend: anti-human CD45-APC (BioLegend Cat# 304011, RRID:AB_314399), anti-human CD45-Brilliant Violet 421™ (BioLegend Cat# 304032, RRID:AB_2561357), anti-human CD4-APC (BioLegend Cat# 300514, RRID:AB_314082), anti-human CD4-PE/Cyanine7 (BioLegend Cat# 300512, RRID:AB_314080), anti-human CD8-FITC (BioLegend Cat# 980908, RRID:AB_2888883), anti-human CD8-Brilliant Violet785™ (BioLegend Cat# 344739, RRID:AB_2566201), anti-mouse CD45-APC (BioLegend Cat# 103111, RRID:AB_312976), anti-mouse CD45-Brilliant Violet 711™ (BioLegend Cat# 103147, RRID:AB_2564383), anti-mouse/human CD11b-Brilliant Violet 570™ (BioLegend Cat# 101233, RRID:AB_10896949), anti-mouse/human CD11b-PE (BioLegend Cat# 101208, RRID:AB_312791), anti-mouse/human CD11b-PE/Cyanine7 (BioLegend Cat# 101215, RRID:AB_312798), anti-mouse Ly6G-FITC (BioLegend Cat# 127606, RRID:AB_1236494), anti-mouse Ly6G-PerCP/Cyanine5.5 (BioLegend Cat# 127616, RRID:AB_1877271), anti-mouse Ly-6C-PerCP/Cyanine5.5 (BioLegend Cat# 128012, RRID:AB_1659241), anti-mouse F4/80-PE/Cyanine7 (BioLegend Cat# 123114, RRID:AB_893478), anti-mouse F4/80-PE (BioLegend Cat# 123110, RRID:AB_893486).

Peripheral blood mononuclear cells (PBMCs) were analyzed using flow cytometry as previously described.^[66] Mice were deeply anesthetized, and blood was collected via cardiac puncture. Whole blood sample was incubated with red blood cell lysis buffer at room temperature for 10 min. Following a centrifugation at 400g, 4 °C, for 5 min, PBMCs were harvested, washed twice with PBS, and centrifuged at 400g, 4 °C for another 5 min. Cells were resuspended in 200 µL flow cytometry staining buffer. Nonspecific binding to Fc receptors was blocked by incubation with a CD16/32‐specific antibody (Biolegend, 101302) at 4 °C for 15 min. Cells were washed and stained with following antibodies: anti‐mouse CD45 brilliant violet 711 (Biolegend, 103147), anti‐mouse CD11b PE (Biolegend, 103147), anti‐human CD3 Brilliant violet 786 (Biolegend, BDB563918), anti‐mouse B220 PE‐Cyanine5.5 (Biolegend, 35045282), anti‐mouse Ly6C FITC (Biolegend, 128006), anti‐mouse mouse Ly6G Pacific blue (Biolegend, 127612), and anti‐mouse F4/80 PE‐Cyanine 7 (Biolegend, 123114). Flow cytometry was performed on an LSRII (BD) and analyzed with FlowJo v 10.4.