Fig 1: Cell culture medium supplemented with replacement serum (RS) does not interfere with detection of proteases(A) Quantification of secreted proteases and inhibitors in cell culture supernatants of M(M-CSF), M(LPS/IFN-γ), and M(IL-4) cultured in media supplemented either with RS or FBS compared to media without cells. Quantified by Western Blot (for TIMP-1 and A2M) or gelatin in gel zymography (for MMP-2, MMP-9). #; sample loaded onto gel was concentrated 25-fold compared to other samples to enhance visualization of gelatin breakdown. Each data point represents cells from a different donor (n = 4). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗p < 0.05; ∗∗p < 0.01; ns non-significant).(B) Quantification of proteolytic activity of M(M-CSF), M(LPS/IFN-γ) and M(IL-4) cultured in media supplemented either with RS or FBS compared to media without cells. Proteolytic activity was measured by a substrate-based degradation assay. The fluorogenic peptide Mca-PLGL-Dpa-AR-NH2 was used to measure proteolytic activity of MMPs, Cathepsin D and Cathepsin E. Each data point represents cells from a different donor (n = 6). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).