Toxicity assays - Mitochondrial assays
9 important questions on Toxicity assays - Mitochondrial assays
What are different ways to inhibit mitochondrial function? (3 examples)
- Binding to cardiolipin (fosfolipide in mitochondria)
- Inhibition of the citric acid cycle
- Uncoupling
What is the JC-1 assay?
It exists in two forms:
- Monomeric form: Emits green fluorescence (~530 nm)
- Aggregated form: Emits red fluorescence (~590 nm)
The potential is decreased when mitochondrials are damaged (less negative). Therefore less aggregation.
How is the JC1 assay conducted?
- Microscopic analyses
- FACs analysis
- Plate reader
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What will you measure when there is mitochondrial stress?
To which cellular mechanisms does the seahorse look?
- OCR = Oxygen Consumption Rate
- ECAR = Extracellular Acidification Rate => lactate production + CO2 production by the mitochondria
- Soms ook PER = Proton Efflux Rate
Of what consists a seahorse graph?
- Basal respiration
- Blocking of ATP-ase synthesis by oligomycin (blocks complex 5) => decrease in oxygen consumption rate because not used for ATP production.
- Adding FCCP => the protons that are still stuck in the inner membrane are pumped back into the matrix by FCCP. Hierdoor verlies je de potentiaal zonder dat er ATP gecreeerd is.
- Hierdoor gaat het dus compenseren => maximal respiration to recover.
- Stopping the ETC completely with rotenone/antimycin => you end up with non-mitochondrial respiration.
Why do you still need rotenone/antimycin when you already add an product that causes proton leaks?
Explain shortly what happens with the OCR during the proces.
Besides antimycin and rotenone, what is another molecule that inhibits the etc?
Blocks complex IV.
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