Fluorescence at excitation and emission wavelengths of 485 and 530 nm, respectively, was measured with a microtiter plate reader (Tecan). Statistical methods Statistical analyses were carried out with SigmaPlot 12. Results are presented as mean ± standard deviation Elacridar price (SD). To enhance the comparability of the assays,

the results were normalized to the average value of the solvent controls (SC) and are expressed as percent change or fold change relative to the SC. Prior to conducting statistical analyses, all data were checked for normality and homogeneity of variance using the Kolmogorov-Smirnov and Levene’s test. A one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used to determine treatments that differed significantly from the SC for data fulfilling the parametric assumptions. Otherwise, the non-parametric Kruskall-Wallis test followed by Dunn’s post hoc test was used. For the detection of significant differences in cytotoxicity assays, the

t test following square root transformation was performed. Differences were considered significant at p < 0.05. Results Cytotoxicity Neutral red retention assay An NR80 value (concentrations resulting in 80% viability of the RTL-W1 cells) of 2.1 mg/L was obtained for the biocide TCC (Figure  2). The exposure of cells to MWCNT at concentrations ranging between 0.78 and 50 mg/L and to the mixture of CNT and TCC (0.39 to 25 mg CNT/L +1% TCC; 3-deazaneplanocin A percentage relative to CNT concentration) did not result in cytotoxicity. Figure 2 Cytotoxic effects of TCC in the NR assay. Cytotoxicity of TCC BYL719 supplier assessed in the neutral red retention assay with RTL-W1 cells. Dots represent the mean of three independent exposure experiments with three internal replicates and are given in percent of the viability of the control. The whiskers show the standard deviation of the Glutathione peroxidase mean; PC, positive control (3,5-dichlorophenol);

SC, solvent control (EtOH); the dashed line marks the threshold of 80%. Concentrations of TCC in the subsequently ROS assay were kept below 0.5 mg/L, i.e., below the NR80 value of 2.1 mg/L. MTT assay In addition to the testing of RTL-W1 cells, cytotoxicity was assessed for T47Dluc cells and H295R cells in the MTT assay. All concentrations of MWCNT (0.5 to 50 mg/L), TCC (31.25 to 500 μg/L), and the mixture of both substances (1.56 mg CNT/L + 15.6 μg TCC/L to 25 mg CNT/L + 250 μg TCC/L, i.e., CNT + 1% TCC) did not result in cytotoxicity in T47Dluc cells (data not shown). The results of the MTT cell viability assay with H295R cells are presented in Figure  3. The percentage of viable cells relative to the ethanol (EtOH) control is plotted against the respective sample concentration. The highest concentration of TCC (500 μg/L) revealed cytotoxicity after the exposure to H295R cells.