The effect of AgNPs on cell viability of a developing and mature cortical culture. To assess the effect of AgNPs on cell viability at the early stages of neuronal development, freshly isolated rat cortical cells were cultured either in the absence (control) or presence of AgNPs at doses ranging from 1 μg/ml to 50 μg/ml. The cell viability/cytotoxicology assay was performed on day 3. The number of live and dead cells in randomly chosen areas of 1 mm2 were counted using the imageJ program. The representative images are shown in A-E and the statistical data of cell viability is presented in the bar graphs of F. Live cells are represented by the green fluorescence of calcein labeling of cell cytosol and neurites, while dead cells are represented by the red fluorescent ethidium homodimer-1 indicating membrane damage. A-F reveals that when cells were cultured in the presence of AgNPs, their viability and axonal outgrowth was decreased by AgNPs in a concentration-dependent manner. To study how AgNPs affected cell viability at more developed stages, neurons and glia were first cultured in control medium for 4 days (G) or 10 days (H) respectively, and both groups of cells were subsequently exposed to various concentrations of AgNPs for another 2 days. The Live-dead cell assay was performed 2 days later. G-H show that AgNPs at concentrations greater than the 5 μg/ml significantly reduced cell viability in both culture conditions. Statistical significance was determined using ANOVA one-way analysis of variance. Post hoc analysis was conducted using Tukey’s test. * P < 0.05. ** P < 0.01. *** P < 0.001. Error bars indicate SEM for all figures. Scale bar, 25 μm.