Victor4 plate reader

To determine the effects of compounds on cell proliferation, cells were plated in 96-well plates at a density of 5000–10,000 cells per well to achieve 70% of confluence in a volume of 100 µL, and grown for 24 hr prior to treatment. Cells were then treated with DMSO or compounds at concentrations ranging from 1.5 nM to 10 µM (threefold dilutions). After 6 days, cell proliferation was measured using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) and Victor4 plate reader (Perkin Elmer, Waltham, MA). Percent inhibition was calculated relative to DMSO signal, and all results shown are the mean of triplicate measurements.
For synergy analyses of drug combinations, 5000–10,000 cells suspended in 100 µl media were plated in triplicate into each well of 96-well tissue culture plates and grown for 24 hr prior to treatments. Ceritinib and CGM097 at different concentrations defined by a dose matrix were added to the cells such that all pair-wise combinations as well as the single agents were represented. Cells were incubated for 72 hr following compound addition, and cell viability was measured using the CellTiter-Glo luminescent cell viability assay (Promega) and Victor4 plate reader (Perkin Elmer). Isobolograms and combination indices were determined as described by Lehar et al. (Lehár et al., 2009 (link)).

A4573 and TC32 cells were seeded into black poly-lysine-coated 96-well plates at 2 x 10³ cells/well and grown for 24 h in serum-free medium (Corning, Union City, CA). Cells were subjected to treatments (Table 1). After 48 h 10 μM, dichlorofluorescein diacetate (DCFA) (Sigma, St. Louis, MO) was added singly or together with the ROS scavenger 5 mM N-acetyl-cysteine (NAC) (Sigma, St. Louis, MO). Cells were incubated at 37°C and 5% CO₂ for 30 min to permit dye uptake and hydrolyzation. Excess dye was removed by washing with PBS. Fluorescence emission was measured in a Victor4 plate reader (Perkin-Elmer, Waltham, MA) at an excitation of 480nm and emission of 535nm. Data represent the mean of three independent measurements ± SE.

Cells were plated into black 96-well plates (1.5 × 10⁵ cells per well) and rested overnight at 37 °C. Cells were incubated for 45 min in the dark with 100 μl of Hanks' balanced salt solution (HBSS) containing HEPES, probenecid (2.5 mM) and Fluo4-NW (20 mM, Invitrogen). In some experiments, cells were pre-incubated with R406 (0.5 μM) or U73122 (3 μM) for 1 h before addition of Fluo4-NW. Fluorescence was monitored before and after the injection of HBSS, LPS (1 μg ml⁻¹) or ATP (1 mM) using a Victor4 plate reader (Perkin Elmer; excitation, 485 nm; emission, 535 nm). All experiments were performed at 37 °C. Fluorescence (F) was normalized against the baseline acquired at the time of the stimulation (F0).