To determine the viability of cells exposed to MWNT-7, we performed an AB assay (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cells were incubated for 24 h at 37 °C in 0.1 ml of culture medium with various concentrations of MWNT-7 in 96-well culture plates.
The control cells were cultured in the culture medium containing dispersant. Viable cells metabolized the dye, which resulted in an increase in the fluorescence intensity, as determined by excitation/emission at 550/600 nm on a fluorescence multiplate reader (PowerScan 4, DS Pharma Biomedical, Osaka, http://www.selleckchem.com/products/Trichostatin-A.html Japan). Cytotoxic activity was calculated as follows: percent cytotoxicity = 100 × experimental value/control value. Test media were assayed 6 times. To determine the effect of endocytosis inhibitors, cells cultured on 96-well culture plates for 24 h were pretreated with chlorpromazine hydrochloride (20 μM; Nacalai) dissolved in PBS or indomethacin (50 μM; SIGMA, St. Louis, MO, USA) dissolved in ethanol for 15 min. The
cells were then exposed to MWNT-7 (50 μg/ml) with the inhibitors for 2 h. The cells were washed learn more twice with Dulbecco’s PBS (DPBS) at 4 °C and cultured in each medium without MWNT-7 or the inhibitors for 22 h. Thereafter, the cells treated with the AB reagent were assayed. Cells were cultured on ibiTreat dishes (μ-dish35 mm high; ibidi GmbH, Martinsried, Germany) for 24 h in a 5% CO2 incubator. The cells were then incubated with or without MWNT-7 (1 μg/ml) for 24 h. Prior to observation, the cells were washed twice and stained with bisbenzimide H33342 fluorochrome
trihydrochloride (H33342, Oxaprozin 1 μg/ml; Nacalai) for 30 min. The cells were visualized using differential interference contrast (DIC) and fluorescence by fluorescence microscopy (AxioObserverZ1, Zeiss, Jena, Germany) in a 5% CO2 chamber at 37 °C using a 40× objective. To determine the effect of endocytosis inhibitors, cells cultured on ibiTreat dishes for 24 h were pretreated with 2 types of endocytosis inhibitors for 15 min and then exposed to MWNT-7 (10 μg/ml) and H33342 for 2 h. The cells were washed twice with DPBS at 4 °C and observed in each medium without MWNT-7 or the inhibitors. We previously have reported that certain cytokines as secreted as part of the inflammatory response in BEAS-2B cells exposed to MWCNTs (Tsukahara and Haniu, 2011). Although the secretion of interleukin (IL)-6 and IL-8 was shown to increase upon exposure to MWCNTs, other cytokines (IL-12, TNF-α, IL-10, and IL-1β) were not detected. Therefore, we selected IL-6 and IL-8 for evaluation in this study. Cytokines in the culture supernatant were measured using a cytometric bead array flex set system (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocol.