How to choose the best mitochondrial assay for your experiments
Every year an appreciable percentage of pharmaceutical drugs are withdrawn from the market due to adverse effects not discovered during preclinical and clinical testing.
Toxicity of drug compounds can lead to mitochondrial damage and dysfunction which have been associated with the pathology of many common diseases.
Mitochondrial assays are an essential part of the drug discovery process, saving time and money by preventing attrition in later clinical studies.
Our assays for assessing mitochondrial toxicity provide a complete solution for all stages of in vitro mitochondrial safety analysis and measurement of key parameters of mitochondrial function. We’ve put this guide together to demystify the selection process and help you find the right assay for your experiments.
What do you need to consider when choosing a mitochondrial assay?
You can first screen mitochondrial health with our ranges of biogenesis and membrane potential assays. If these pick up adverse indicators, you can use our oxidative phosphorylation (OXPHOS) assays to investigate energy impairment and discover what’s going on in greater detail.
Our assays can help you look at:
- Mitochondrial membrane potential
- Mitochondrial biogenesis
- Superoxide levels
- Real-time mitochondrial tracking
- Oxidative phosphorylation via Complex I, II, III, IV and V activity
- Antioxidant activity
How do I measure mitochondrial biogenesis?
Mitochondrial biogenesis assays measure the rate at which cells generate new mitochondria, providing a measure of mitochondrial health.
How our mitochondrial biogenesis assays can help
Our assays measure biogenesis by measuring the ratio of mitochondria and nuclear-encoded proteins, making them the ideal tool to uncover chronic effects of mitochondrial DNA replication and protein synthesis. Our MitoBiogenesis™ kits come with everything you need to run your assay, including pre-measured dyes and buffers.
MitoBiogenesis™ In-Cell ELISA Kit (Colorimetric)
- Inhibition of mitochondrial biogenesis by chloramphenicol assessed by monitoring the relative amounts of COX-I (mitochondrial DNA encoded) and SDH-A (nuclear DNA encoded)
- Suitable for adherent cell samples
- Colorimetric detection
MitoBiogenesis™ In-Cell ELISA
- IRDyes® (ab110216)
- Colorimetric (ab110217)
- Fluorescent (ab140359)
How do I measure mitochondrial membrane potential ?
Mitochondrial membrane potential is a key indicator of mitochondrial activity levels. Our wide range of assay kits enables you to measure membrane potential whatever your application.
How membrane potential assays can help
A number of fluorescent dyes are available that accumulate in mitochondria due to their membrane potential. Our wide range of assay kits can help you measure membrane potential in the majority of applications.
JC-10 Mitochondrial Membrane Potential Assay Kit (Microplate)
- Robust method for monitoring mitochondria membrane potential changes
- In normal cells, JC-10 forms red fluorescent aggregates, but changes to a monomeric form and stains cells green in apoptotic and necrotic cells
- Superior water solubility compared to JC-1
TMRE-Mitochondrial Membrane Potential Assay Kit
- TMRE is a cell-permeant, positively-charged, red-orange dye
- TMRE accumulates in active mitochondria due to their relative negative charge
- Suitable for microplate readers, flow cytometry, and fluorescent microscopy
JC-10 Mitochondrial Membrane Potential Assay Kit
Flow cytometry (ab112133)
Microplate reader (ab112134)
How do I investigate energy impairment?
Changes to oxygen consumption or membrane potential may indicate energy impairment upon drug treatment. To identify the exact cause of energy impairment, activity of metabolic enzymes- including oxidative phosphorylation complexes and enzymes involved in wider mitochondrial metabolic pathways like the TCA cycle- should be assessed.
How our OXPHOS assays can help
To investigate the in vitro activity of individual oxidative phosphorylation (OXPHOS) enzyme complexes, we use immunocapture to enable specific biochemical assays for each complex.
Our in vitro MitoTox™ assays measure the effect of drug treatment on oxidative phosphorylation complex activity in a colorimetric microplate format that can be easily adapted for high-throughput assessment. The assays can be used to test IC50 of two compounds in a dose-response format, and up to 23 compounds at a single concentration. MitoTox™ assays are available for each of the OXPHOS complexes.
MitoTox™ Complex II + III OXPHOS Activity Assay
- Tests the direct inhibitory effect of compounds on Complex III activity in just 30 minutes
- Colorimetric readout at 550 nm
- Intra- and inter-assay variation <10%
Complex I Enzyme Activity Microplate Assay Kit (Colorimetric)
- Complex I enzyme activity assay kit
- Plates are pre-coated with highly specific capture antibodies
Mitochondrial Complex III Activity Assay Kit
- Measures the absorbance of reduced cytochrome C at 550 nm.
- Fast and reliable method for determining the activity of complex III
- Sensitive up to 10 mU/ml; linear up to 30 mU/ml
When should I quantify oxidative stress?
A thorough investigation of oxidative stress should be conducted where initial screens show increased ROS production to identify its exact cause.
How our assays can help
We’ve developed effective tools to measure ROS production by direct ROS measurement, quantification of ROS-induced protein modifications, and measurement of antioxidant capacity. Many of our kits are available in 384-well formats to allow high-throughput experiments, letting you get more done in the same amount of time.
DCFDA / H2DCFDA - Cellular ROS Assay Kit
- Complete kit including standard curve
- Compatible with fluorescence microscopy, flow cytometry and fluorescence plate reader.
- Highly cited in
700 publications
GSH/GSSG Ratio Detection Assay Kit II (Fluorometric - Green)
- Fast, easy protocol for measuring reduced and oxidized glutathione (GSH/GSSG) in plasma, urine, tissue and cell extracts
- High sensitivity; detection limit as low as 1 pmol/100 µL
- Available in 96-well or 384-well microtiter-plate formats; easily adapted to automation without a separation step
Glutathione Peroxidase Assay Kit (Colorimetric)
- Compatible with urine, cell cultures, tissues, blood and other biological fluids
- Quick, reliable results in 40 minutes
- High sensitivity of ~ 0.5 mU/ml of GPx in samples
