Mitochondria are the powerhouses of the cell, responsible for producing ATP through oxidative phosphorylation (OxPhos) while also playing key roles in cellular signalling, apoptosis, and metabolic regulation. Beyond their well-known function in energy metabolism, mitochondria are now recognised as central players in human health and disease, with mitochondrial dysfunction implicated in cancer, neurodegenerative disorders, metabolic diseases, inflammation, and ageing.
Cellular respiration is correlated with ATP synthesis. The rate of OxPhos increases in response to higher cellular energy demand (generating ATP), which consumes oxygen. In cells, the metabolic signalling that generates ATP (such as glycolysis, amino acids catabolism and beta-oxidation), along with those cellular functions that consume ATP (proliferation, autophagy, biosynthesis etc.) can be reflected by the alterations of oxygen consumption rate (OCR). Therefore, OCR is a widely informative readout of cellular metabolic functions. BioAscent provides the most advanced and reliable approach to measuring OCR in either intact cells or isolated mitochondria with the state-of-the-art Seahorse XF Pro platform – a microplate-based technology that enables simultaneous monitoring of OCR and the extracellular acidification rate (ECAR), a measure of energy production shift from OxPhos to glycolysis. This system allows us to investigate mitochondrial function in response to pharmacologic inhibitors/effectors or genetic interventions in a robust and high-throughput fashion.
In practice, several modifications can be made to enhance our understanding of mitochondrial perturbations:
Uncoupler Challenges: Seahorse XF is an in vitro assay and does not fully replicate the in vivo environment. As a result, cellular responses to energy demands or pharmacological/genetic interventions may be masked under basal conditions. The addition of uncouplers such as FCCP or BAM15 can push cells into a more energetically demanding state, revealing these hidden effects.
Real-Time Stimulation: Physiologically relevant stimuli can be administered directly within the Seahorse XF assay to assess cellular metabolic adaptation. For example, anti-CD3 and anti-CD28 antibodies can be used to activate T cells, while norepinephrine can stimulate brown adipocytes or mature cardiomyocytes. This approach allows for real-time measurement of shifts between oxidative phosphorylation (OxPhos) and glycolysis.
Media Optimisation: Careful manipulation of Seahorse XF assay media is crucial for investigating specific signalling mechanisms or metabolic targets. For instance, selectively providing oxidizable substrates such as glucose, glutamine, pyruvate, or galactose enables targeted analysis of metabolic pathways.
Get in touch to discover how BioAscent can tailor Seahorse XF Pro assays to address specific scientific questions in your mitochondrial research projects.
At BioAscent, we integrate three powerful Agilent platforms to help you enhance immune cell potency, fate, and persistence:
xCELLigence RTCA: Measure immune cell-mediated killing in real time
Seahorse XF Pro: Discover and tune immune cell programming to mount a sustained and robust antitumor response
NovoCyte flow cytometer: Monitor T cell expansion, compound toxicity, drug effects on tumour cell growth, immune cell behaviour under different conditions
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At BioAscent, we offer comprehensive mitochondrial biology services, combining expertise in biochemistry, live-cell imaging, and functional metabolic assays to support drug discovery. Our state-of-the-art platforms enable high-resolution analysis of mitochondrial function, facilitating the development of novel mitochondrial-targeted research and therapeutics. With our cutting-edge metabolic profiling, we support investigations into mitochondrial metabolism in cancer, immunology, neurodegeneration, and metabolic disease, providing tailored assays and expert guidance to accelerate your research.
Contact us to find out more about mitochondrial biology services from BioAscent.
