Bioactivity of Mu-IFNαA translated from bicistronic vectors. In conditioned medium from mesenchymal stem cell (MSC) clones where both bioactivity and immunoreactivity were measured, the specific activity of interferon in each medium was calculated and each clone was separately color-coded. In all cases, the activity was determined by comparison with a standard murine interferon, produced in Escherichia coli and purified to homogeneity, and the mean of each group of values are shown with a horizontal line. (a) Left: conditioned medium from six monoclonal MSC lines stably transfected with (from left to right) pEF3-MuIFNαA, pEF3-MuIFNαAEMCV*ChFP and pEF3-MuIFNαAcmycChFP were assayed for bioactivity by the antiviral assay. Right: in a separate experiment, conditioned medium from 12 monoclonal MSC lines stably transfected with pEF3-MuIFNαA and from four monoclonal MSC lines stably transfected with pEF3-MuIFNαAEMCVChFP were assayed. (b) Conditioned medium from six monoclonal MSC lines stably transfected with pEF3-MuIFNαAEMCV*ChFP (left) and pEF3-MuIFNαAcmycChFP (right) were assayed for Mu-IFNα immunoconcentration by ELISA. (c) Specific activities of the Mu-IFNαA within each conditioned medium were calculated by dividing the bioactivity by the immunoconcentration. Bacterial recombinant Mu-IFNαA purified to homogeneity has a specific activity of 5 × 107 to 10 × 107 units/mg; this range is indicated with the brackets between the two datasets. ChFP, monomeric cherry fluorescent protein.