IFN-γ protects articular cartilage from GAG depletion mediated by IL-1β activated RA FLS ex vivo. Full depth articular cartilage explants were obtained from bovine limbs (BACE). Two 12-well plates were set up in parallel, one with RA FLS the second without RA FLS. At the beginning of each experiment, one BACE was place in each well. BACE (± RA FLS) were incubated in DMEM/F12 or medium containing; 0.1 ng/ml IL-1β, 10 ng/ml IFN-γ or IL-1β with IFN-γ for 72 hours when cartilage explants and supernatants were reserved. Three RA FLS cell lines were tested; cytokines (alone or in combination) were examined in duplicate. Cartilage depletion at end point was visualised in Safranin-O/Fast Green-stained sections. Representative images from one experiment are reported (A-F), reduced intensity of red stain denotes proteoglycan loss (original magnification × 20). A-C BACE cultured without RA FLS, D-E corresponding BACE from RA FLS co-cultures which had been incubated in DMEM/F12 medium containing: 0.1 ng/ml IL-1β (A and D), 10 ng/ml IFN-γ (B and E) or IL-1β with IFN-γ (C and F) for 72 hours. The depth of GAG depletion (μm) in each BACE, measured from the articular surface to the red/orange tidemark (denoted by black block arrow) is presented graphically in (G); mean ± SEM for three experiments reported. MMP-3 was measured by ELISA in culture supernatants harvested from each well after 72 hours in culture. (H) MMP-3 (ng/ml); mean ± SEM for three experiments. One-way analysis of variance was used to analyze differences in GAG depletion and MMP-3 levels: *P < 0.05 and **P < 0.0001.