To analyze the intrinsic surface expressions of IL-17 or TNF receptors, FLS were stained using APC-conjugated anti-human CD217 (IL-17Ra; Invitrogen), BV421 anti-human CD120b (TNF Receptor Type II; BD Biosciences), and mouse anti-human CD120a (TNF Receptor Type I; BD Biosciences) with FITC-conjugated anti-mouse IgG secondary antibody (BD Biosciences).
FLS cultures were incubated with serum-free media for 5 h and then stimulated with recombinant IL-17 and TNF-α for 1 h. Culture media was changed, and the cells were incubated with FITC-conjugated anti-human vascular cell adhesion protein 1 (VCAM1; BD Biosciences), PE-Cy™5-conjugated anti-human intercellular adhesion molecule 1 (ICAM1; BD Biosciences), and PE-Cy™7-conjugated anti-human neural cell adhesion molecule 1 (NCAM1; BD Biosciences). To detect ROS levels, cells were stained with MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed with a FACSCantoII flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star).
FLS cultures were incubated with serum-free media for 5 h and then stimulated with recombinant IL-17 and TNF-α for 1 h. Culture media was changed, and the cells were incubated with FITC-conjugated anti-human vascular cell adhesion protein 1 (VCAM1; BD Biosciences), PE-Cy™5-conjugated anti-human intercellular adhesion molecule 1 (ICAM1; BD Biosciences), and PE-Cy™7-conjugated anti-human neural cell adhesion molecule 1 (NCAM1; BD Biosciences). To detect ROS levels, cells were stained with MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed with a FACSCantoII flow cytometer (BD Biosciences), and data were processed with FlowJo software (Tree Star).