Figure 4: Magnetofection enhances viral vector-mediated gene delivery in C2C12 myoblasts and myotubes: C2C12 myoblasts and fully differentiated myotubes were incubated with either naked recombinant adenoviral vectors (RAd-GFP or RAd-IGF-1) or RAd+MNPs complexes (RAd+PEIMag2) and exposed to an optimal magnetic field for 30 minutes. To assess gene delivery efficiency, GFP expression was evaluated using microscopy imaging and fluorescence quantitation 48 hours after infection for both groups. In a similar manner, IGF-1 levels were measured in the culture medium by radioimmunoassay. A Representative microscopy images of myoblasts (upper panels) and myotubes (lower panels) infected with RAd-GFP and RAd- GFP+MNPs at 1.5 and 2.5 fgFe/VP MNP-to-Viral Particle ratios. After imaging, cells lysates were used to perform quantitation of GFP expression. Comparison between conventional viral transduction and magnetofection was plotted as absolute B and C and relative-to-naked virus fluorescence levels B’ and C’. Unspecific signal from cells alone, MNPs and lysis buffer were subtracted to each group previous to data analysis. To assess magnetofection efficacy with RAd-IGF-1, culture medium from myotubes was collected 48 hs after gene transfer protocols and processed for IGF-1 extraction and quantitation. Comparison between conventional viral transduction and magnetofection was plotted as absolute D and relative-to-naked virus concentration D’. IGF-1 basal levels in myotubes alone, as well as unspecific readings from extraction buffer, were subtracted to both experimental groups in order to show only viral vectormediated IGF-1 production. In a subset of myotubes, IGF-1 expression was analyzed in the culture medium at days 2 (D2), 4 (D4) and 6 (D6) after gene delivery and compared between non-infected, transduced with naked virus and magnetofected cells. Plots show absolute E and relative-to-myotubes alone E’ IGF-1 levels for each time point. *Statistically significant difference with p < 0.05 between non-infected myotubes and naked virus or magnetofection groups. #Statistically significant difference with p < 0.05 between naked virus and magnetofection groups. Data values represent mean ± S.E.M. n=3 for GFP groups, n=4 for IGF-1 at 48 hs and n=6 for IGF-1 longitudinal analysis. fgFe/VP=femtograms of iron per Viral Particle