Celltiter glo luminescence cell viability assay

After 48 h of spheroid formation, chemotherapeutic agents, namely fluorouracil combined with epirubicin and cyclophosphamide (FEC) and docetaxel combined with doxorubicin and cyclophosphamide (TAC) were administered to the spheroids in clinically relevant combinations at the peak plasma concentrations as described previously [26 (link)]. Galectin-1 was applied in a concentration of 30 μg/ml. Medium (untreated) and solvent controls were included in each experiment. Solvents used to control the effect of the drugs were 0.2%H2O plus 0.26 % NaCl for FEC therapy, 0.01 % H2O plus 0.21 % NaCl for TAC treatment and 0.15 % phosphate buffered saline (PBS) for galectin-1. Each treatment and control was performed in six replicates. The drugs were allowed to take effect for a total of 48 h. Chemotherapeutics were obtained from the pharmacy of the University Hospital LMU (Munich, Germany). Treatment efficacy was assessed using an ATP assay (CellTiter-Glo® Luminescence Cell Viability Assay, G8461, Promega, Germany) to quantify cell survival in vitro. Mean cell survival was expressed as percent of residual metabolic activity relative to the solvent controls.

LHC9 and LHC basal medium, trypsin, PBS and ProLong® Gold antifade reagent with DAPI were purchased from Life Technologies (Saint Aubin, France). Co₃O₄ particles (Co₃O₄P) were supplied by Merck (Fontenay Sous Bois, France) and, according to the manufacturer’s quality control sheet, their purity was of 98.4 %. CoCl₂ x 6 H₂O and Polystyrene Latex Beads (LB-3 in aqueous suspension; 0.3 μm mean diameter size) were purchased from Sigma-Aldrich (Lyon, France). CellTiter-Glo® Luminescence Cell Viability Assay and CellTiter-Blue® Assay were purchased from Promega (Charbonnieres, France). All other reagents were purchased from Sigma-Aldrich.

10,000 cells were plated into each of the wells of a black 96-well plate. Treatments were given 24 h after plating. After 72 h from plating, the Cell Titer Glo Luminescence Cell Viability Assay (Promega, Madison, WI, USA) was used to determine the cell viability of each of the experiments. The results of the plates were interpreted by using a 96-well plate reader. The averages of the intensity units were calculated and plotted onto a graph corresponding to the concentration of drug given using GraphPad Prism. For the EC50 studies, the averaged intensity units were normalized to percentages and plotted onto a graph with the corresponding concentration of drug given using GraphPad Prism.