Fc block clone 2.4g2

For flow cytometric analysis and cell sorting, tumors, lymph nodes and spleens were collected from mice and digested with 0.26 U/ml Liberase TM and 0.25 mg/ml DNase I at 37°C for 30 min. Samples were then filtered through a 70 μm cell strainer and washed twice with staining buffer. Cells were re-suspended in staining buffer (PBS with 2% FCS and 0.5 M EDTA). Cells were incubated with Fc Block (clone 2.4G2; BioX Cell) for 10 min. Subsequently, specific antibodies were added and staining was continued for 30 min on ice. Information for all the antibodies used are provided in Supplementary Table 1. OT-I specific T cells were stained using iTAg Tetramer/H-2K^bOVA (SIINFEKEL) (MBL). After a washing step, cells were either analyzed on a BD Fortessa (BD) or sorted by AriaIIIu (BD). For the staining of cathepsins, splenocytes were stained with CD11c, B220, MHCII, CD8 and CD11b and then fixed with 4% PFA (Biolegend) for 30 minutes. Fixed cells were then washed twice with the 1x intracellular staining perm and wash buffer (Biolegend). Antibody against CTSA, CTSB, CTSD or CTSH was added and incubated overnight respectively. Alexa Fluor 568 Goat-anti-Rabbit IgG was added as the secondary antibody. CD11c⁺MHCII⁺B220⁻ was gated and the expression of cathepsins was evaluated by the fluorescence intensity. Analysis of flow cytometry data was performed using Flowjo (Treestar).