Oxygen Consumption Assay [HS Method] is a highly flexible 96 or 384-well fluorescence plate reader-based approach, for the direct, real-time analysis of cellular respiration and mitochondrial function. The easy-to-use MitoXpress® Xtra assay allows measurement of extracellular oxygen consumption rates (OCR) with whole cell populations (both adherent and suspension cells), isolated mitochondria, permeabilised cells and a wide range of 3D cultures including: tissues, small organisms, spheroids, scaffolds and matrixes. The assay is also suitable for measurement of isolated enzymes, bacteria, yeasts and moulds. In this assay, MitoXpress® Xtra is quenched by O2 through molecular collision, and thus the amount of fluorescence signal is inversely proportional to the amount of extracellular O₂ in the sample. Rates of oxygen consumption are calculated from the changes in fluorescence signal over time. The reaction is non destructive and fully reversible (neither MitoXpress® Xtra nor O₂ are consumed), facilitating measurement of time courses and drug treatments. Luxcel’s flexible plate reader format, allows multiparametric or multiplex combination with Luxcel’s other products, as well as combining with commonly available reagents to measure glycolysis, LDH, JC-1, MMP (Ѱ), ROS, and cellular ATP. For example, MitoXpress® Xtra in combination with Luxcel’s pH-Xtra® – Glycolysis Assay allows the simultaneous real-time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and the shift (flux) between the two pathways under pathological states. Plate Preparation 3D RAFT cultures were prepared with either A549 or HepG2 cells at the indicated density in 240ul DMEM / Collagen solution on a 96-well plate. RAFT cultures were formed as per manufacturer’s protocol. Oxygen Consumption Measurements. For oxygen consumption measurements MitoXpress®-Xtra stock was prepared in 16ml of pre-warmed DMEM and culture media was replaced in each well with 150µl of this solution. Where applicable, 1µl of compound stock (150X) was added to each well. Wells were then sealed by overlaying with 100µl pre-warmed HS mineral oil to inhibit oxygen back diffusion into the sample. This is best done using a repeater pipette. The plate was then measured kinetically on a FLUOstar Omega (BMG Labtech) for 90-120mins with ~2 minute interval exciting the probe at 380nm and measuring emission at 650nm. Ratiometric measurements were performed using the following delay and gate settings. Delay 1: 30s, Gate 1: 30s, Delay 2: 70s, Gate 2: 30s. Extracellular Acidification Measurements. Three hours prior to measurement the RAFT culture plate was placed in a CO2 FREE incubator at 37°C, 95% humidity, in order to remove CO₂ from the plate material. Spent media was removed and 2 wash steps were performed using the Respiration Buffer (0.5 mM KH2PO4, 0.5 mM K2HPO4, 20 mM Glucose, 4.5 g/L NaCl, 4.0 g/L KCl, 0.097 g/L MgSO4, 0.265 g/L CaCl2), finally 150µl of Respiration Buffer containing pH-Xtra probe at the recommended concentration was added to each well. The plate was then measured kinetically on a FLUOstar Omega (BMG Labtech) for 90-120mins with ~2 minute interval exciting the probe at 380nm and measuring emission at 615nm. Ratiometric measurements were performed using the following delay and gate settings. Delay 1: 100s, Gate 1: 30s, Delay 2: 300s, Gate 2: 30s.Oxygen consumption provides detailed information on mitochondrial function, specifically on the activity of the electron transport chain (ETC),while extracellular acidification (ECA) informs on glycolytic flux. Measurements are conducted on standard 96-well microtitre plates, on a fluorescence plate reader, and facilitate a deep insight into the metabolic behaviour of the 3D culture and into how metabolism is perturbed by a particular compound or environmental condition. 3D cell culture facilitates the development of complex intra-cellular interactions thereby helping to narrow the gap between in vitro and in vivo biological systems. Adoption of 3D technologies has however been limited, in part due to difficulties associated with producing reproducible 3D cultures. Difficulties can also arise due to an incompatibility with certain in vitro assay technologies.

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