Pe conjugated anti mouse cd25

To determine the number of regulatory T cells in the splenocytes, flow cytometry was performed, as previously described [7 (link)]. The splenocytes were washed with PBS and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA) and phycoerythrin (PE)-conjugated anti-mouse CD25 (eBioscience, San Diego, CA, USA). Next, the cells were fixed and stained with Alexa Fluor 647 anti-mouse Foxp3 (BD Biosciences, San Jose, CA, USA) overnight at 4 °C in the dark. After washing, the cells were analyzed. In order to measure Th1 and Th17 cells in the splenocytes, FITC-conjugated anti-mouse CD4 (eBioscience, San Diego, CA, USA), PE-conjugated anti-mouse IFN-γ (eBioscience, San Diego, CA, USA), and PerCP/Cyanine5.5-conjugated anti-mouse IL-17A (eBioscience, San Diego, CA, USA) were stained according to the supplier’s instructions.

In order to evaluate the phenotype of different immune cell populations, cells derived from the spleen or tumor of vehicle- or AZD1480-treated mice were stained with the following antibodies: phycoerythrin (PE)-Cy7-conjugated anti-mouse CD3 (BioLegend), Alexa Fluor 700 (AF700)-conjugated anti-mouse CD4 (BD Biosciences), AF647-conjugated anti-mouse CD8 (BioLegend), Horizon V450-conjugated anti-mouse CD45 (BD Biosciences), peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated anti-mouse CD4 (BD Biosciences), PE-conjugated anti-mouse CD25 (eBioscience), AF700-conjugated anti-mouse CD127 (eBioscience), AF647-conjugated anti-mouse CD11c (BioLegend), PE-conjugated anti-mouse CD11b (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD86 (BD Biosciences), allophycocyanin (APC)-H7-conjugated anti-mouse CD80 (BD Biosciences), FITC-conjugated anti-mouse CD11b (BD Biosciences), AF647-conjugated anti-mouse Ly6G (BioLegend) and PECy7-conjugated anti-mouse Ly6C (BioLegend).