Celltiter 96 aqueous mts reagent

DLD1 cells were seeded in a 96-well plate at a density of 2 x 10⁴ cells/well. Immediately after seeding, the cells were transfected with mirVana™ miRNA mimic has-miR-34a-5p (Invitrogen; ThermoFisher Scientific, USA) or mirVana™ miRNA mimic Negative control (Invitrogen; ThermoFisher Scientific, USA), according to the manufacturer’s instructions. The transfection mixture consisted of Lipofectamine RNAiMAX transfection reagent (8 μl/ml; Invitrogen; ThermoFisher Scientific, USA), Opti-MEM^TM (Invitrogen; ThermoFisher Scientific, USA), and micro-RNA or negative control at a concentration of 50 nM was added to the cells and incubated for 48 h. After the transfection, E2 at a concentration of 100 nM was added to the medium for 24 h. Next, the cells were incubated with 10 μM of MTS reagent according to the manufacturer’s instructions (CellTiter 96^® Aqueous MTS Reagent, Promega, USA). The absorbance was measured at a wavelength of 490 nm (Epoch2, BioTek instruments, USA).

BMDCs were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate in 180 μl per well of complete RPMI 1640 cell culture media. After incubation for a few hours, cells were treated with an increasing concentration of PMG5 (50, 100, 200, 400, 800, and 1600 μg/ml). Also, corresponding concentrations of soluble PAMAM G5 (6.25, 12.50, 25, 50, 100, and 200 μg/ml) were tested. Plates were then incubated at 37°C with 5% CO₂ for 48 hours. Next, Cell Titer 96 aqueous MTS reagent [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, purchased from Promega Corporation, San Luis Obispo, CA] was added to the cells following the manufacturer’s instructions (63 (link), 68 –70 (link)). After incubation for 2 hours, cell viability (%) was calculated on the basis of the absorbance measured at 490 nm using a SpectraMax Plus spectrophotometer (Molecular Devices, San Jose, CA).

Human colorectal adenocarcinoma DLD1 cells were purchased from the American Type Culture Collection (ATCC). HCC827GR (gefitinib‐resistant lung cancer) cells were kindly provided by Dr. Pasi A. Jänne at the Department of Medical Oncology, Dana‐Farber Cancer Institute, Boston, MA (Engelman et al., 2007). Colon cancer DLD1 cells and gefitinib‐resistant lung cancer HCC827 cells (HCC827GR) were grown in RPMI‐1640 supplemented with 10% FBS. All cells were maintained as monolayer cultures at 37°C in an incubator with 5% CO₂. DLD1 and HCC827GR cells were seeded in a 96‐well plate at a density of 5 × 10⁴ and 1 × 10⁵ cells per each well, respectively. After a 24‐hr incubation, various concentrations of arctiin, arctigenin, and bioconversion samples in glucose‐deprived RPMI‐1640 were added and incubated for another 24 hr. Cell viability was determined by performing 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) assays using the CellTiter 96 AQueous MTS Reagent (Promega). Twenty microliters of MTS solution was added to each well, and the cells were further incubated at 37°C for 30 min in an atmosphere containing 5% CO₂. Next, the absorbance was measured using a microplate reader (Molecular Devices) at 490 nm and at 650 nm as the reference absorbance.

Cells were plated at 3 × 10³ cells per well in 96-well plates. For RNAi experiments, cells were transfected with siRNA 24 h after cell plating. The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay was performed using the CellTiter 96^® Aqueous MTS reagent (Promega, Madison WI), according to the protocol provided by the manufacturer. The experiment was performed five times. For cell counting, cells were trypsinized and counted using the trypan blue exclusion method to quantify cell viability.