In vitro Matrigel tube formation assay. A) Matrigel tube formation assay using growth factor-reduced Matrigel (BD Biosciences) was performed. The test substances used were recombinant human Id1 (rhuId1) (1 and 10 nM), basic fibroblast growth factor (bFGF) (60 nM, R&D Systems, positive control) and phosphate-buffered saline (PBS) (negative control). The treated human dermal microvascular endothelial cells (HMVECs) (1.8 × 104 cells/400 μl) were plated on Matrigel in the presence of Id1, bFGF or PBS for six hours at 37°C. Photographs (100×) were taken and tubes were counted by a blinded observer. Tubes were defined as elongated connecting branches between two identifiable HMVECs. A) HMVECs formed tubes to Id1 at 10 nM (the concentration for peak HMVEC chemotactic activity for HMVECs, n = number of independent experimental replicates). B) Rheumatoid arthritis (RA) synovial fluid (SF) was pre-incubated with mouse anti-human Id1 antibody (Abcam) for two hours at 4°C. RA SFs were added in the Protein A agarose beads and were rotated at 4°C overnight. The next day, the samples were spun down and SFs collected. SFs were diluted 1:100 with PBS. We then measured Id1 in the SFs pre- and post-Id1 neutralization, and as shown, effectively eliminated Id1 from the SFs. RA SF depleted of Id1 showed less HMVEC tube forming activity compared to “sham”, IgG depleted SFs. Photographs (100×) were taken and tubes were counted by a blinded observer (n = number of independent experimental replicates).