Apc conjugated anti mouse cd4 antibody

Fresh tumor tissues were cut into small pieces and were incubated in a combination of 0.15% type I collagenase (Sigma-Aldrich; catalog no. C0130-500MG) and 0.15% type II collagenase (Sigma- Aldrich; catalog no. C6885-1G) at 37 °C for 1 h. Digested tissues were filtered with a 100-μm cell strainer, followed by a 70 μm cell strainer. Single cells from each sample after centrifugation were stained on ice for 15 min with an Alexa Fluor 647–conjugated anti-mouse F4/80 antibody (123122, BioLegend), an APC–conjugated anti-mouse CD3 antibody (17-0032-80, BioLegend), an APC–conjugated anti-mouse CD4 antibody (100411, BioLegend), and a PerCP–conjugated anti-mouse CD8 antibody (100731, BioLegend). The stained samples filtered with a 40-μm cell strainer were scanned on a FACScans (Becton Dickinson) and data were analyzed with a CellQuestPro software (BD Biosciences). The gating strategy of flow cytometry is presented in Supplementary Fig. 11.

Lymphocytes from spleens were resuspended in staining buffer (PBS containing 3% of fetal bovine serum) and stained for 30 minutes at 4°C with an APC/Cy7-conjugated anti-mouse CD3 antibody (Lot. 100330, BioLegend), PE/Cy7-conjugated anti-mouse NK-1.1 antibody (Lot. 108714, BioLegend), APC-conjugated anti-mouse CD4 antibody (Lot. 100412, BioLegend), PerCP-conjugated anti-mouse CD8a antibody (Lot. 100732, BioLegend) as well as their corresponding isotype control antibodies as a negative control. Cells were fixed and permeabilized using the fix/perm kit (Lot. 88-8823-88, eBioscience). Intracellular IFN-γ and IL-4 staining was performed on the cells stained with a PE-conjugated anti-IL-4 antibody (Lot. 5014104, BioLegend) and FITC-conjugated anti-IFN-γ antibody (Lot. 505806, BioLegend) and its isotype control antibodies as a negative control. Flow cytometry was performed on a BD Canto II flow cytometer (BD Biosciences).