, 1997). The data was fitted using Curve Expert v1.3 (D. Hyams, Hixson, TN, USA). The rate of reduction of resazurin to resorufin is taken as a GDC-0449 research buy measure of cell viability. Therefore, βV is the cytotoxic potency estimate for the particles in the CTB assay, with negative values indicating a decrease of reduction rate and positive

values indicating an increase of reduction rate of resazurin. Reduction (increased fluorescence), oxidation and hyper-reduction (decreased fluorescence) of assay reagents (resazurin or resorufin) by nanomaterials will bias the cytotoxic potency estimates. Specifically, reduction of resazurin by the nanomaterial will result in underestimate of cytotoxicity, while decrease of fluorescence of resorufin by oxidation or hyper-reduction will lead to an overestimation of cytotoxicity. Therefore, change in fluorescence in the acellular assay, under otherwise identical conditions as the cellular assays, was fitted to Eq. (1) to estimate the magnitude of the interference (βINT). Unbiased cytotoxic potency estimates (βV-INT) was obtained from equation(2) βV-INT=βV-βINTβV-INT=βV-βINT Data were analyzed using two-way or three-way Analysis Of Variance (ANOVA) on data relative to control (0 μg/cm2 dose). Where the assumptions of normality and equal variance were not satisfied, transformations on ln or rank were conducted

prior to analysis. Holm–Sidak multiple comparisons procedure was performed to elucidate AZD6244 manufacturer the patterns of significant effects (α = 0.05). The statistical analyses are presented within figure legends. The pattern of effects presented in Fig. 1 could be interpreted as a decrease of CTB reduction after exposure of the A549 (left panel) and J774A.1 (middle

panel) cells to CNTs for 24 h. However, an identical pattern of fluorescence can be observed in absence of cells (right panel). That the decrease of fluorescence was unrelated to cytotoxicity, but was due rather to physical quench of photons was obvious from the settling of CNTs after 10 min or 1 h in the assay with cells (Fig. 2A) or without cells (Fig. 2B). However, interference was not limited to physical quench. only When reduced reagent (resorufin) from spent A549 supernatant was incubated with CNTs followed by clarification by centrifugation, a trend of a decrease of fluorescence was observed for CNT-2 and CNT-4, at the highest dose of 100 μg/cm2 (Fig. 3). This loss of fluorescence was accompanied visually by decrease of pink color intensity. The modified assay, consisting of clarification by centrifugation prior to reading at 10 min and 2 h, resolved the major issues of interference by physical quench in A549 cells (Fig. 4A) and J774A.1 cells (Fig. 4B). Difference in potency of the various CNTs, SiO2 nanoparticles and micron-sized TiO2 and SiO2 is reflected in significant Particle x Dose interaction, two-way ANOVA (A549 cells, p = 0.002; J774A.1 cells, p = 0.004).