For flow cytometry, Zombie Violet™ Fixable Viability Kit (BioLegend) or 7AAD Viability Kit (BioLegend) were used as a live/dead marker. The surface stained with the following antibodies obtained from BioLegend: the matching isotype controls, APC/Cyanine7 anti-mouse CD45 Antibody, PE-anti-mouse CD3e, FITC anti-mouse CD4, PE/Cy7 anti-mouse CD8a, Alexa Fluor® 647 anti-mouse CD279 (PD-1), APC-anti-mouse CD11b, FITC-, Percp/Cyanine-anti-mouse-F4/80, PE-anti-mouse CD86, PE-anti-mouse CD206. The intracellular staining cells were firstly permeabilized after surface staining with Intracellular Staining Perm Wash Buffer (BioLegend) for 20 min according to the manufacturer's instructions and then stained with Alexa Fluor® 647 anti-mouse/human Ki-67, Alexa Fluor® 647 anti-mouse FOXP3.
For flow cytometry of RAW264.7 cells or BMDMs, the CD11b positive and F4/80 positive cells were firstly identified and gated by the expression of CD86 or CD206. Tumor tissues were excised and made into single cell suspensions, the CD45 positive cells were initially identified and gated by the expression of CD4 and CD8, PD1, Ki67, or FOXP3. All samples were performed by the FACS Canto II (BD Biosciences) or CytoFLEX (Beckman Coulter) cytometers. Data were further analyzed by FlowJo V10 software.
For flow cytometry of RAW264.7 cells or BMDMs, the CD11b positive and F4/80 positive cells were firstly identified and gated by the expression of CD86 or CD206. Tumor tissues were excised and made into single cell suspensions, the CD45 positive cells were initially identified and gated by the expression of CD4 and CD8, PD1, Ki67, or FOXP3. All samples were performed by the FACS Canto II (BD Biosciences) or CytoFLEX (Beckman Coulter) cytometers. Data were further analyzed by FlowJo V10 software.