Apc conjugated anti mouse cd3 antibody

Tumor-infiltrating T cells were analyzed by flow cytometry. First, tumor tissues were excised on day 27 after tumor re-challenge and digested in collagenase (1 mg/mL in RPMI; Sigma-Aldrich) for 2 h at 37 °C with gentle stirring. Red blood cells were lysed and the resulting tumor cell suspension was passed through a 40-μm strainer. The cells were then stained with the following fluorescent antibodies: APC-conjugated anti-mouse CD3 antibody (BioLegend), PE-conjugated anti-mouse CD4 antibody (BioLegend), and PerCP/cyanine5.5-conjugated anti-mouse CD8a antibody (BioLegend). The percentage of CD3(+)CD4(+) and CD3(+)CD8(+) cells was analyzed using a BD FACSCalibur flow cytometer, and CellQuest Pro and FlowJo software v10.0.7 (BD Bioscience).

For FACS analysis of mouse renal cells, single-cell suspensions were stained with antibodies at 4 °C for 30 min. The following antibodies from Biolegend were used: BV421-conjugated Zombie (423114), APC/Cy7-conjugated anti-mouse CD45 antibody (103116), APC-conjugated anti-mouse CD3 antibody (100236), FITC-conjugated anti-mouse CD4 antibody (100406), PE/Cy7-conjugated anti-mouse CD8 antibody (100722), PE-conjugated anti-mouse B220 antibody (103208), PE/Cy7-conjugated anti-mouse CD31 antibody (102417), PE-conjugated anti-mouse GP38 antibody (127407).

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.