Pe conjugated isotype control

For flow cytometry, cells were detached using Alfazyme (PAA Laboratories), washed, and incubated in 50 μl of 3% (v/v) FCS in phosphate-buffered saline (PBS) with labeled antibody (phycoerythrin [PE]-conjugated) anti-TNFR1, anti-TNFR2, or the respective PE-conjugated isotype control (R&D Systems) for 45 min on ice in the dark. For the quantification of TACE/ADAM17 surface densities, cells were incubated with an antibody detecting the TACE ectodomain (MAB9301, R&D Systems) followed by the incubation with a PE-conjugated secondary antibody (Biolegend). After antibody staining, cells were washed three times and measured in the PE channel of a FACS Canto II (Becton Dickinson). For detection of TNFα binding, cells were incubated with TNFα-Biotin and Streptavidin-FITC or let unstained. Cells were washed three times and measured in the FACS Canto II FITC channel. Histograms were generated by the use of FlowJo (Tree Star).

Standard single color cell surface staining with PE-conjugated isotype-control or anti-ADAM10 or anti-ADAM17 mAb (R&D Systems) was performed as previously described 22 (link), 25 (link).