Celltiter96 non radioactive cell proliferation kits

Normal PmECs and breast cancer cells were seeded onto 96-well plates at a density of 1 × 10⁵ cells/mL, and left overnight to attach. Culture medium was removed and the cells were treated with serial dilutions of lutein (0.5–2.0 μM) in fresh culture medium with 10% FBS, or cultured in medium with DMSO (in the same concentration as carotenoid samples) as a control. Cell viability was determined after 48 h by MTT assay using CellTiter96® Non-Radioactive Cell Proliferation kits (Promega, Madison, WI, USA), in which the yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by mitochondrial dehydrogenase in viable cells to purple, insoluble crystals of formazan. Cells were incubated for 4 h with MTT solution at 37 °C and formazan crystals were solubilized in a lysing buffer overnight at room temperature (RT). The product was quantified by measurement of absorbance at a 570 nm wavelength with the use of a BioTek Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA). All experiments were performed in triplicate and yielded similar results. Results are representative of an average of three independent experiments. Data are presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which was assumed as 100%.

ARPE-19 cells were seeded in 96-well plates at a density of 2 × 10³ cell/well and cultured in 100 μL medium overnight for attachment. Then, the culture medium was removed and the cells were treated with serial dilutions of carotenoids (0.5–2.0 μM) in fresh culture medium with 10% FBS or cultured in a medium with DMSO as a control. Cell viability was determined after 48 h by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using the CellTiter96^® Non-Radioactive Cell Proliferation Kits (Promega, Madison, WI, USA), in which the yellow tetrazolium salt is reduced by mitochondrial dehydrogenase of viable cells to purple, insoluble crystals of formazan. Cells were incubated for 4 h with MTT solution at 37 °C. Then, formazan crystals were solubilized in lysing buffer overnight at room temperature (RT). The reaction product was quantified by measurement of absorbance at a 570 nm wavelength using a BioTek Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). All experiments were performed in triplicate. Results are representative of an average of 3 independent experiments. Data are presented as proportional viability (%), by comparing the treated group with the untreated cells, the viability of which is measured to be 100%.

The cell toxicity to SWCNTs-Ag and pSWCNTs-Ag was determined using Cell Titer 96® Non-Radioactive cell proliferation kits (Promega, Madison, WI). A549 (human lung carcinoma), Hep2 (hepatocellular carcinoma) cells and J774 (murine macrophages) cell lines were used for the cytotoxicity assay. The assay is a colorimetric assay based on the reduction of the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide). As per the manufacturer’s protocol, 1 × 10⁴ cells/well in 100 μL of minimum essential medium-10 (MEM-10, Gibco, Life technologies, Grand Island, NY) were seeded into a 96-well plate. After overnight incubation at 37°C in 5% CO₂ humidified atmosphere, the media from the 96-well plates were replaced with the MEM-10 media containing 62.5, 31.25, 15.12 or 7.8 μg/mL of either SWCNTs-Ag or pSWCNTs-Ag. The treated cells were further incubated at 37°C under 5% CO₂ for 24 and 48 h. At the end of the each incubation period, 15 μL of MTT dye were added into each well and the plate was allowed to incubate again for the next 4 h, in darkness. The reaction was then stopped using 100 μL of the stop solution. The absorbance of the plate was measured at 570 nm using a TECAN Sunrise™ enzyme-linked immunosorbent assay (ELISA) plate reader (Tecan US, Inc., Morrisville, NC, USA). Non-treated cells, in growth media, were used as a control.