αcd16 32 antibody

Isolated TIL from GL261-OVA tumors were blocked with αCD16/32 antibody (Biolegend) and stained with PE/APC H-2K^b-SIINFEKL dextramer (Immudex) according to the manufacturer’s staining protocol. TIL were subsequently stained with fixable viability dye eFluor 780 (1:1000, Thermo Fisher, 65-0865-14), CD45-BV510 (clone 30-F11, Biolegend), CD11b-PE/Dazzle594 (clone M1/70, Biolegend), CD3-FITC (clone 17A2, Biolegend), and CD8-PercpCy5.5 (clone 53-6.7, Thermo Fischer). TIL were analyzed on a FACSAria II system (BD Bioscience).

To measure cell surface expression of CD120a (TNFR1), CD120b (TNFR2), and CD11b, BMDMs were replated on Day 4 at a density of 22,000/cm² in a six‐well format. On Day 7, cells were stimulated as indicated. At harvest time point, cells were placed on ice, washed in the plate with cold PBS, harvested by gentle scraping in cold PBS, and passed through a 40 μm cell strainer. After Fc blocking for 15 min using α‐CD16/32 antibody (BioLegend 101301, 1:50), each sample was split to be incubated with APC α‐CD11b (BioLegend 101212, 1:333), APC‐α‐CD120a (BioLegend 113005, 1:100), or biotin α‐CD120b (BioLegend 113403, 1:100) for 45 min. The α‐CD120b‐stained sample was further incubated with APC‐Strepavidin (BioLegend 405207, 1:500) for 10 min. 7AAD (BioLegend 420404, 1:100) was added as a viability dye. Flow cytometry was performed on CytoFLEX flow cytometer (Beckman Coulter). Data were analyzed using FlowJo v10. Median fluorescence intensity (MFI) of unstained control was deducted from MFI of stained samples before calculation of relative expression levels. For measuring cell surface expression of CD120a after TNFR1 siRNA transfection, cells were replated on Day 4 in a 12‐well format, transfected with siRNA 24 h before the experiment, and analyzed using α‐CD120a antibody as above.