ELISA
Fibroblasts were immortalized by transfection using a simian virus 40
large (SV40) T antigen gene-containing plasmid and then cultured in DMEM
(GIBCO) supplemented with 10% FCS. Fibroblasts (1 x
105) from healthy control, and from patients with
IL17RA- [3] or IL17RC- [7] deficiency, or
ACT1T536I/T536I [12], or our patient were
stimulated with IL-17A (100 ng/ml), TNF-α (20 ng/ml), alone or in
combination, or IL-1β (10ng/ml) (R&D, 317-ILB, 210-TA, 201-LB), or left
unstimulated. After 24 hours, levels of GRO-α in the supernatants were
measured by ELISA (R&D, DY275) following manufacturer’s instructions.