Fig. 4.
Id1 is contained in fibroblast exosomes. Fibroblasts from RA, osteoarthritis (OA), and normal (NL) STs were plated and maintained in CMRL medium supplemented with 20 % FBS, 2 mM L-glutamine, and 1 % penicillin/streptomycin in T175cm2 flasks and were used between passages 4 and 10. Cultures were moved to serum-free media DMEM/F-12 with Peprogrow serum replacement (Peprotech) for 2 days before harvesting. Fibroblast culture supernatants were concentrated to 1 mL (per flask) by centrifugation through an Amicon Ultracel 30 K filter (EMD Millipore, Billerica, MA, USA). Exosomes from cell supernatants were isolated and purified by serial ultracentrifugation. Nanoparticle Tracking Analysis was used to quantify the size and concentration of particles within the exosome fractions. a and b Cells were spun out at 1500 rpm for 5 min. Then the supernatants were cleared of heavier debris by spins at 10,000 × g for 30 min and 30,000 × g for 1 h. Exosomes were then spun down at 110,000 × g for 4–20 h. Exosome pellets were washed in PBS at 110,000 × g for 1.5 h – overnight. Some exosomes were further purified using a density gradient Optiprep (Sigma-Aldrich). Optiprep was diluted with PBS to produce the following layers: 5, 10, 15, 20, 30, 40, and 50 % w/v (densities of 1.031, 1.050, 1.084, 1.110, 1.163, 1.215, and 1.268 g/mL). The expected size of exosomes is between 25 and 135 nm and >70 % of the EVs isolated fall into this range (red area). c Representative diagram showing that we find more exosomes in RA (yellow and blue areas) compared to OA (blue area only) in fibroblast supernatants. d and e Whole and lysed (0.5 % Triton X-100) exosome fractions were then measured for Id1. We found that >80 % of the detected Id1 is contained within RA fibroblast cell supernatant exosomes. We similarly found that OA and RA SF have exosomes containing Id1 (n = number of sample wells measured for exosomes isolated from SF specimens from two separate patients for OA; and three separate patients for RA). All data was pooled in the respective OA and RA SF groups. Id1 inhibitor of DNA binding 1, OA osteoarthritis, RA rheumatoid arthritis, SF synovial fluid