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Microplate-based assays to measure O2 consumption or glycolytic flux in live cells without the need for specialized equipment.
A fluorescent-based approach for direct real-time analysis of cellular respiration and glycolytic flux.
|Assay||What it measures||How it works||Assay kits|
|Extracellular Oxygen Consumption (OCR) Assay||O2 consumption rate||As cell respiration lowers O2 concentration, dye fluorescence increases|
|Intracellular Oxygen Assay||Intra-cellular O2 levels||Dye fluorescence is inversely proportional to oxygen concentration||ab197245|
|Glycolysis Assay (ECAR)||Extracellular acidification (glycolysis) rate||Lactate causes extracellular acidification, dye fluorescence increases|
|Fatty Acid Oxidation Assay||O2 consumption rate on blocking of sugar metabolism||As fatty acid oxidation lowers O2 concentration, dye fluorescence increases|
Case study: Measurement of mitochondrial metabolism in cultured cells
Oxygen consumption rate and extracellular acidification assays can be used together to build a metabolic picture of response to drug treatment.
We measured oxidative phosphorylation and anaerobic glycolytic flux in the human hepato-carcinoma cell line HepG2 (Figure 1). Cells were treated with FCCP (an OXPHOS uncoupler) and antimycin A (a complex III inhibitor). Cellular energy production was monitored by measuring ATP content.
Figure 1: HepG2 cells (seeded at 6.5 x 104 cells/well) were treated with 1 µM antimycin A and 2.5 µM FCCP. Oxygen consumption (white column), extracellular acidification rate (black column) and ATP concentration (stripped column) data are shown as percentage of untreated control. All measurements were performed on a FLUOstar Omega (BMG Labtech).
As expected, complex III inhibition by antimycin A reduced oxygen consumption rate to undetectable levels, while FCCP treatment significantly increased oxygen consumption. Both antimycin A and FCCP treatment caused a dramatic increase in extracellular acidification due to the increase in lactate production. Cellular ATP concentration was virtually unchanged in treated cells as energy production is maintained.
In conclusion, we have described how to study changes to glycolytic and oxidative metabolism in response to mitochondrial modulators in real-time with a simple microplate-based assay.