There are several applications that require the measurement of oxygen consumption or glycolytic pathway routing in living cells, but cannot accommodate special tools for the purpose. This need is met by microplate-based assays.
This approach is built upon fluorescent detection to enable the direct real-time assessment of cell respiration and the flow through the glycolytic reactions.
Key features of microplate-based assays are:
- They are based on fluorophores that respond to changes in oxygen or pH
- The reactions are non-destructive and can be turned back completely, which means timelines and drug treatments can be evaluated and measured
- No specialized probes or any other tools are required, as the assay measures the fluorescent signal using a standard fluorescent reader or a TR-F plate reader.
||What it measures
||How it works
|Extracellular Oxygen Consumption (OCR) Assay
|As cell respiration lowers O2 concentration, dye fluorescence increases
|Intracellular Oxygen Assay
|Dye fluorescence is inversely proportional to oxygen concentration
|Glycolysis Assay (ECAR)
||Extracellular acidification (glycolysis) rate
||Lactate causes extracellular acidification, dye fluorescence increases
Core assay: ab197244
Glycolysis stress test kits: ab222945 and ab222946
|Fatty Acid Oxidation Assay
|O2 consumption rate on blocking of sugar metabolism
||As fatty acid oxidation lowers O2 concentration, dye fluorescence increases
Companion kit to OCR assay: ab217602
Complete kit: ab222944
Case Study: Examining the Metabolism of Mitochondria within Cells in Culture
While studying how the body metabolism reacts to treatment with a drug, the rate at which oxygen is consumed can be considered, along with assays for acidification outside the cell, to get a fuller picture.
In this experiment, the human hepato-carcinoma cell line HepG2 was studied for oxidative phosphorylation and the flow of glucose through the anerobic glycolysis pathway, as shown in Figure 1. The cells were first mixed with an uncoupler of oxidative phosphorylation, namely, FCCP, and a complex III inhibitor, namely, antimycin A. Afterwards, the production of energy was monitored by looking at the ATP content in the cell.
The Extracellular Oxygen Consumption Assay was used to measure the oxygen consumption after placing a mineral oil seal in place to keep ambient oxygen from diffusing back into the solution.
The glycolytic flux was evaluated using the Glycolysis Assay [Extracellular Acidification]. Here, unsealed samples were necessary as otherwise, the carbon dioxide would have contributed towards increased acidification of the extracellular space.
A Luminescent ATP Detection Assay was used to find the ATP concentration. Unsealed samples were used to measure the ATP content after ECA measurements were carried out.
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 the researchers hypothesized, when antimycin A was added to inhibit complex III, the rate of oxygen consumption dropped to undetectable levels, but rose after the sample was treated with FCCP.
Both the added chemicals resulted in a sharp elevation in the acidification outside the cell as a result of greater lactate production. The cellular ATP content remained almost the same after treatment with either of these, showing that the production of energy was conserved at a steady level.
This experiment describes the study of changes in oxygen consumption and glycolytic flux when molecules that modulate mitochondrial function were added to the sample, using a real-time microplate assay.
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