What Does Oligomycin Do In Seahorse Assay?

Oligomycin inhibits ATP synthase (complex V), and is injected first in the assay following basal measurements. It impacts or decreases electron flow through the ETC, resulting a reduction in mitochondrial respiration or OCR. This decrease in OCR is linked to cellular ATP production.

How does the Seahorse assay work?

The Agilent Seahorse XF ATP Real-Time rate assay measures and quantifies the rate of ATP production from glycolytic and mitochondrial system simultaneously using label-free technology in live cells. In mammalian cells, glycolysis and oxidative phosphorylation (OXPHOS) pathways provide the majority of cellular ATP.

How does FCCP increase OCR?

The mitochondrial uncoupler FCCP increased OCR (A, B), due to increased aerobic metabolism, and increased ECAR (C, D), due to increased glycolysis consequent to decreased mitochondrial ATP generation, with a net effect of decreased cellular ATP levels (E).

How does Seahorse measure ATP?

The new Seahorse XF Real-Time ATP rate assay utilizes XF technology to detect mitochondrial Oxygen Consumption Rates (OCR) and glycolytic Extra-Cellular Acidification Rates (ECAR). These data are then transformed to mitoATP and glycoATP production rates using well-validated algorithms.

How does Seahorse Bioanalyzer work?

Seahorse XF Analyzers measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a multi-well plate, interrogating key cellular functions such as mitochondrial respiration and glycolysis.

What is Oligomycin seahorse?

Oligomycin inhibits ATP synthase (complex V), and is injected first in the assay following basal measurements. It impacts or decreases electron flow through the ETC, resulting a reduction in mitochondrial respiration or OCR. This decrease in OCR is linked to cellular ATP production.

How does Oligomycin inhibit ATP synthase?

Oligomycin (Omy) is an inhibitor of ATP synthase by blocking its proton channel (Fo subunit), which is necessary for oxidative phosphorylation of ADP to ATP (energy production). The inhibition of ATP synthesis also inhibits respiration.

How does Oligomycin affect oxygen consumption?

As expected, the sequential addition of oligomycin resulted in significant inhibition of oxygen consumption. Experiments with digitonin-permeabilized T98G cells indicated that BKA and CAT (2.5 μM of each, B, D) were effective in fully inhibiting ADP-stimulated oxygen consumption.

Does FCCP increase ATP production?

FCCP has a protonophore effect that will dissipate the proton gradient, thereby reducing ATP synthesis, as noted in our results.

Does FCCP block or decrease ATP production?

FCCP Decreases the Levels of ATP in PC12 Cells.
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How is oxygen consumption measured seahorse?

Oxygen consumption rate (OCR) is measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. Initially, baseline cellular OCR is measured, from which basal respiration can be derived by subtracting non-mitochondrial respiration.

Why do we use 36 ATP instead of 38?

In eukaryotic cells, the theoretical maximum yield of ATP generated per glucose is 36 to 38, depending on how the 2 NADH generated in the cytoplasm during glycolysis enter the mitochondria and whether the resulting yield is 2 or 3 ATP per NADH.

How is mitochondrial ATP production measured?

Measurement of intracellular ATP in living cells Mitochondrial ATP production and its cytosolic accumulation can be evaluated by either perturbing Ca²⁺ resting conditions, with the generation of changes in intracellular Ca²⁺ concentration triggering ATP synthesis, or analyzing the contribution of the single oxidative

What causes extracellular acidification?

Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO₂, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate^– + H⁺ and the export of products into the assay medium is the source of glycolytic acidification.

What is the purpose of Bioanalyzer?

The Bioanalyzer can be used to assess the size and quality of DNA over a range of sizes and concentrations. The DNA 1000 assay can be used with extracted, clean DNA to look at the size profile of a sample from 25bp to 1000bp at a concentration of 0.1-50ng/ul.

What is the difference between Bioanalyzer and TapeStation?

A: The Agilent 2200 TapeStation system can run anywhere from 1 to 95 samples. The Bioanalyzer is “chip- based” and can only fit 11 or 12 samples per run. Therefore, the service fee for Bioanalyzer run is charged on a per chip basis. Both Bioanalyzer and TapeStation can run cDNA or NextGen library samples.

What is oligomycin used for?

Oligomycin has been used to probe tumor cell dependence on glycolytic metabolism,¹⁷⁶ the effect of F₁F₀-ATPase inhibition and ATP depletion on tumor necrosis factor-α (TNF)-induced apoptosis,^177,178 and the role of oxidative phosphorylation on the activation of BAX and Bak tumor suppressors.

What is the mode of action for oligomycin?

Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase.

What does oligomycin do to mitochondria?

Oligomycin prevents the increase in mitochondrial respiration induced by ADP without inhibiting uncoupler-stimulated respiration. NADH remains high and NAD⁺ is too low for the citric acid cycle to operate.

Does oligomycin have a direct effect on ATP synthase?

Oligomycin has been recognized as a potent inhibitor of the mitochondrial ATP synthase since 1958 when it was reported by Henry Lardy et al. (1).

Is oligomycin an uncoupling agent?

Here we show that oligomycin induces proton uncoupling subsequent to inhibition of ATP synthesis, as evidenced by recovery of O₂ uptake to near baseline levels. Uncoupling is uniquely rapid and readily observed in HepG2 cells but is also observed at longer times in the unrelated H1299 cell line.