5% fetal bovine serum (FBS) according to the methods details in Maletz et al. [84]. T47Dluc cells were cultured at 37°C, 7.5% CO2, and maximum humidity. H295R cells The human adrenocarcinoma cells (H295R) were obtained from the American Type learn more Culture Collection (ATCC; Manassas, VA, USA) and were grown in 75-cm2 flasks with 8 mL supplemented medium at 37°C with a 5% CO2 atmosphere as described previously [73, 85]. Nanoparticles suspension Test suspensions of 1 to 100 mg/L of MWCNT were prepared by ultrasonication of

the raw material with a microtip (70 W, 0.2″ pulse and 0.8″ pause; Bandelin, Berlin, Germany) in distilled water for 10 min. Transmission electron microscopy (TEM) images showed the presence of small agglomerates and individual nanotubes in the medium (Figure  1). Figure 1 TEM pictures of MWCNT. Agglomerates (A), single nanotubes (B), and tubes sticking out of the agglomerates (C, D) visualized by transmission

electron NSC 683864 manufacturer micrographs of sonicated MWCNT in distilled water. Cytotoxicity assays For determining the effect of particles on cell viability, different assays were used. Potential interferences of MWCNT and the fluorescence measurement were prevented by using black microtiter plates. Neutral red retention assay The neutral red retention (NR) assay was performed according to Borenfreund and Puerner [86] with slight modifications as detailed in Heger et al. [87] by using RTL-W1 cells. Briefly, 4 × 105 cells were seeded into each well (except for the blanks) of a

96-well microtiter plate (Nunc) and directly treated in triplicates with the particle suspensions. To guarantee optimal culture conditions, cells were exposed in a 1:1 mixture of MWCNT suspension or TCC solution and double-concentrated L15-Leibovitz medium, resulting Terminal deoxynucleotidyl transferase in final MWCNT-concentrations of 3.13 to 50 mg CNT/L and TCC concentrations of 7.8 to 10 × 103 mg/L. After incubation for 48 h at 20°C in the dark, the sample solution was discarded, and each well was rinsed with 100 μL phosphate-buffered saline (PBS) to remove any excess medium. One hundred microliters of a 0.005% neutral red solution (2-methyl-3-amino-7-dimethylaminophenanzine, Sigma-Aldrich) was added to each well except for the blanks. After an incubation time of 3 h at 20°C in darkness, the amount of extracted NR was determined by absorption measurement at 540 nm and a reference wavelength of 690 nm using a microtiter plate reader (Infinite M200, Tecan Instruments, Männedorf, Switzerland). Thereafter, concentrations resulting in cell vitality of 80% were calculated and identified as NR80 Akt inhibitor values according to Heger et al. 2012 [87]. For detection of significant differences, the t test following square root transformation was performed using SigmaPlot 12. Results are given as relative values to the untreated control in percent.