Cd11c pacific blue

Cells harvested from the spleen were used for single color and multi-color staining controls. Control and tumor samples were resuspended at a concentration of 10⁶ cells/100 uL in FWB in round bottom polystyrene tubes. Cells were stained with the following antibody fluorophore conjugates: CD45-PerCP/Cy5.5, CD11c-Pacific Blue, CD11b-AF700, MHCII-APC/Cy7, Ly6c-BV510, Ly6g-APC, CD38-FITC (Biolegend, San Diego, CA), F480-PE/Cy7 (Tonbo Bioscences, San Diego, CA), CD206-PE (R&D Systems, Minneapolis, MN) and propidium iodide (PI, Enzo Life Sciences, Farmingdale, NY). All antibody staining took place for 30 min at 4 °C in the dark. For all experiments, cells were acquired on the BD LSRII Fortessa flow cytometer. Compensation and sequential gating was performed with FlowJo software (FlowJo LLC, Ashland, OR). Populations of myeloid cells that were identified included macrophages, dendritic cells, granulocytic myeloid derived suppressor cells (G-MDSC), monocytic myeloid derived suppressor cells (M-MDSC) and M0, M1 and M2 macrophage phenotypes (Supplementary Fig. S1).

Mice were euthanised at the end of the study; single-cell suspensions were prepared from the spleens and tumours as described in our previous study.^{18 (link)} Spleen cells and tumour cells were stained with respective stain cocktails. Anti-mouse CD11b-PE (phycoerythrin) (101208), Ly6C-APC (allophycocyanin) (128015), Ly6G-FITC (fluorescein isothiocyanate) (127605), Gr1-PE/Cy7 (108416), CD11C-Pacific Blue (117321), major histocompatibility complex II (MHCII)-AF700 (107622), CD3-PerCP/Cy5.5 (100217), CD4-PE/Cy5.5 (100410), CD8-BV510 (100751), interleukin (IL)-2-PE (503808), IFN-γ-APC (505809), tumour necrosis factor (TNF)-α-FITC (506304), and Granzyme B-PE/Cy7 (372213) were purchased from Bio Legend (San Diego, CA). Cells were acquired through BD LSRII flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometric analysis was performed by using the Flow Jo software (Tree Star Inc., Ashland, OR, USA). For co-cultures experiments, cells were sorted by using BD FACS ARIA III (BD Biosciences, San Jose, CA, USA).