Cellular accumulation and cytocompatibility analysis of MNPs in A549 cells. A. A549 cells growing on 96-well tissue culture plates were incubated without or with 50 μg/ml MNP or PLGA-MNP for 24 h, washed and fixed. Adherent cells were fluorescently stained with rhodamine-phalloidin (red) to visualize cellular cytoskeleton actin and with Hoechst (blue) to visualize nuclei. Fluorescent or bright-field images acquired using an automated microscope IN Cell Analyzer-1000 are presented in the left or middle panels. Right panels show corresponding magnified images indicated by box in the middle panels. B. A549 cells growing on 96-well tissue culture plates were exposed to 50 μg/ml MNP for up to 24 h and imaged by IN Cell Analyzer-1000. Percentage of cells with accumulated particles were quantified by IN Cell Investigator software and presented. *p < 0.05 compared to untreated control. C. A549 cells growing on 96-well tissue culture plates were incubated with various concentrations (ranging from 10 to 250 μg/ml) of MNP or PLGA-MNP for 24 h, washed and fixed. Cells were treated with 1 μg/ml quantum dots as a toxicity control (+ve control). HCS biocompatibility analysis was performed using IN Cell Analyzer-1000 equipped with Investigator software by quantifying cell adherence to the plates. Values in the plotted line graph are fold change in viable cell numbers ± SEM of three independent experiments in triplicate from five randomly selected fields/well containing at least 300 cells. *p < 0.05 compared to untreated control. D. Real-time electric impedance sensing measurements of A549 cells treated with 100 μg/ml MNP, PLGA-MNP or 10 μg/ml nocodazole (as a toxicity control). Each data point is the mean Cell Index ± SEM of technical triplicates. A representative plot of three independent experiments is shown.