Easy assay analysis

Ann-Cathrin Volz  discusses the simple evaluation of metabolic assays

The 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. The 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. These 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. They 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. The 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 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

The 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.

The 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). The 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).

The ROS-Glo assay detects the production of ROS - H2O2 to be exact. H2O2 produced by the cell leads to the generation of a luminescent signal via the intermediate luciferin. The amount of cellular ROS naturally increases upon treatment with a ROS inducer like Menadione (Fig.2a). Antioxidants like N-Acetyl-L-cysteine (NAC), on the other hand, have the potential to inhibit cellular ROS production. HeLa cells were treated with a high dose (50 µM) of Menadione and on top with increasing concentrations of NAC (12.5 to 400 µM). The measurement of luminescence in the Vantastar revealed a dose-dependent reduction of cellularly produced H2O2 with increasing NAC concentrations (Fig.2b).

BMG Labtech’s Vantastar plate reader is an ideal tool to measure luminescence-based assays with maximum ease of use. The Enhanced Dynamic Range (EDR) function ensures each well is read with the optimal gain and highest sensitivity. This allowed to miniaturise the assay from a 96-well to a 384-well format. Furthermore, the Vantastar automatically applies the ideal focal height and blocks unwanted stray signal from adjacent wells during the detection.

The plate reader enables user-friendly luminescence measurements with significantly reduced error susceptibility. These features combined with the homogenous assay format of the metabolic assay kits (Glucose-Glo, Lactate-Glo and ROS-Glo) represent the ideal combination to analyse cellular metabolism also in a miniaturised format.

Ann-Cathrin Volz is with BMG Labtech

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