Anti tnfa

Mouse blood was retroorbitally collected in heparinized capillary tubes followed by RBC lysis (BioLegend). Mouse tumors were collected after sacrifice and dissociated using 30% collagenase digestion (30 mins, 37°C). Single cell suspensions in 5% FBS in PBS by passing through 40 μm cell strainer were assessed for viability with Trypan Blue and manually counted, then incubated with Fc receptor blocking solution followed by fluorophore-conjugated antibodies.
For cell surface staining, fluorescence-labeled mouse antibodies (including anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-Ly6C, anti-Ly6G, anti-F4/80, anti-CD11c, anti-CD80, anti-CD86, anti-MHCII purchased from BD Biosciences, BioLegend or eBioscience) were added to samples for 30 min at 4 °C in the dark. For further intracellular staining, cells were washed with PBS supplemented with 5% FBS, permeabilized with Permeabilization Buffer (BD Bioscience) and stained with fluorescence-labeled mouse antibodies (including anti-IFNγ, anti-GzmB, and anti-TNFa purchased from BD Biosciences or BioLegend) for 30 min at 4 °C in the dark. Cells were washed and resuspended in 5% FBS in PBS. Flow cytometry was performed on an LSRII (BD Biosciences), and data analyzed using FlowJo Version X (Tree Star).