MICROPLATE READERS
EASY ASSAY ANALYSIS
Ann-Cathrin Volz discusses the simple evaluation of metabolic assays
T
he term cell metabolism describes all the chemical changes that take place in a cell to generate energy and the components for essential processes.
Next to the breakdown of molecules into their basic components and the removal of useless by-products, this also includes the new synthesis of molecules needed for cell growth. Te different molecular conversion steps are catalysed by enzymes and are strongly interconnected. In “metabolic pathways”, the product of one conversion becomes the substrate for another one.
Consumed carbohydrates (saccharose,
starch, etc.) are digested and broken down into monosaccharides such as glucose or fructose in the intestinal tract.
Tese can subsequently be used by cells to generate energy in the cellular glucose metabolism. Under aerobic conditions, monosaccarides undergo glycolysis via the tricarboxylic acid cycle. Here, energy is mainly obtained from NADH, which is used by the electron transport chain in the mitochondria to produce ATP. Under anaerobic conditions, lactate is produced, which can also be used to monitor cellular glucose metabolism. Unlike glucose, reactive oxygen species
(ROS) do not have any direct nutritional value. Tey are intentionally produced by the cell to function as signalling molecules. However, if high ROS levels are built up inside the cell, also referred to as oxidative stress, they can damage DNA, RNA, proteins and lipids.
WHY STUDY METABOLIC PATHWAYS? In biology, scientific research on cell metabolism focuses on different topics such as understanding the fundamental metabolic processes, metabolic changes and their role in diseases, the development of therapies, the identification of unknown metabolic processes and metabolites, etc. Te investigation of the glucose metabolism is especially of growing interest in research areas such as cancer, diabetes, neuroscience and immunology. ROS detection assays are needed particularly in screening campaigns to determine the effect of compounds on enzymes such as the NADH oxidase and their catalysed reactions. Moreover, these assays are used to study the effects of antioxidant therapies. Diagnostic and particularly drug
Fig. 1. (a & b). Luminescent signal generated from starved versus non-starved HeLa cells using the Glucose-Glo and Lactate-Glo assays
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screening approaches using larger libraries set the demand for plate-based metabolic assay formats that enable their analysis in miniaturised formats (with a just a few µL per well) to save reagents, samples and consumables.
ANALYSE ASSAYS WITH EASE Te Glucose-Glo and Lactate-Glo kits (from Promega) provide the simplicity of a homogenous add-mix-measure assay. Cellular consumption of glucose and lactate production can be analysed from conditioned medium mixed with the respective detection reagents. If glucose or lactate is present in the sample, the respective enzymatic cascade is triggered, and a luminescent signal is produced. Te assays were used to monitor glucose consumption of HeLa cells over 48h. Half of the HeLa cells were starved overnight (w/o glutamine and w/o serum). Te luminescence results acquired by BMG Labtech’s Vantastar multi-mode reader show a decreased consumption of glucose by starved cells (Fig.1a). In line with this, starved cells also demonstrated a decreased lactate production (Fig.1b). Te ROS-Glo assay detects the
production of ROS - H2 H2
O2 produced by the cell leads to the O2 to be exact.
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