Modulation of Chondrocyte ECM by RvD1

The human chondrocyte cell line C28/I2 (gift from Mary Goldring, Cornell Medical College, New York, New York, USA; ref. 54 (link)) was utilized for micromass assays as described previously (20 (link)). Micromasses were stimulated with or without IL-1β (30 ng/ml) alone or in combination with 17R-RvD1 (0.1–100 nM) for 24 hours, and ECM accumulation was calculated following staining with Alcian blue as reported (21 (link)). Briefly, micromasses were fixed (4% glutaraldehyde, 15 min) acidified with HCl, and stained with Alcian blue 8GS overnight before dye extraction with guanidine hydrochloride (200 μl, 6M). Absorbance of extracted dye was measured at 620 nm using a Multiskan Bichromatic 348 spectrophotometer and normalized to DNA content using SYBRgreen dye (excitation 485 nm and emission 535 nm) with a TECAN M200 spectrophotometer. These values were used to generate ECM accumulation concentrations and are expressed as percentage change from control micromasses. In some experiments, the FPR2/ALX receptor antagonist WRW₄ (10 μM) was added to micromasses 10 minutes prior to 17R-RvD1 addition, and ECM accumulation was evaluated after 24 hours.