Approaches to measure endpoints Flashcards
(28 cards)
What does the MTT assay measure in cells?
Cell viability, proliferation and cytotoxicity
How does the MTT assay work?
The reduction of MTT (yellow salt) to purple formazin crystals by metabolically active cells
What is the relationship between formazin productions and cell viability in the MTT assay?
The amount of formazin produced is proportional to the number of viable cells
What cellular activity does the MTT assay primarily reflect?
Mitochondrial activity
Name a limitation of the MTT assay related to cell metabolism
Results can be affected by the metabolic state, cell density and medium composition
Name a limitation of the MTT assay related to cytotoxicity
It only measures mitochondrial metabolic activity not direct cell death. Compounds that impair mitochondrial function will reduce MTT signal even in the cells are still alive causing an overestimation of cytotoxicity
Why can the MTT assay itself be cytotoxic?
MTT and the solvents used to dissolve formazin can damage or kill cells during the assay
What is a technical challenge when solubilizing formazin in the MTT assay?
Organic solvents used for solubilisation can cause protein precipitation and light scattering
How can culture conditions influence MTT assay results?
Factors like pH and glucose levels can alter assay outcomes
Why might cells with higher metabolic rates skew MTT assay results ?
The may produce more formazin per cell, overestimating viability
Can the MTT assay distinguish between cell death and reduced metabolic activity?
No, it cannot reliably distinguish between the two
What does the ROS-Glo H2O2 assay measure?
Hydrogen peroxide - a key reactive oxygen species
What is the main application of the ROS-Glo assay
Assessing oxidative stress and ROS generation in cells
What is a key advantage of the ROS-Glo assay?
It is sensitive, rapid, and does not require wash steps (homogeneous)
What can interfere with the ROS-Glo assay’s luminescence signal?
Luciferase inhibitors
What is a detection-related limitation of the ROS-Glo assay
It only measures H2O2 and not other ROS (O2, O, OH)
What is an interference-related limitation of the ROS-Glo assay?
GSH is high in the liver which neutralises H2O2 masking true ROS levels
What does the COMET assay detect?
Overall DNA damage (strand breaks)
Briefly describe how the COMET assay works
Cells are embedded in agarose, lysed and subjected to electrophoresis; damaged DNA migrates forming a comet tail.
What type of toxicity is the COMET assay most sensitive for?
Genotoxicity and DNA repair
Name a limitation of the COMET assay
% of tail is semi quantitative, artefacts from cell handling, tissue with rapid turnover could mask toxin induced effects
What tests could you run after the COMET assay to confirm genotoxicity
AMES test, mammalian cell mutation assay, micronucleus assay
What pathway does the Keratinosense assay meassure?
The antioxidant response element (ARE) dependent pathway in keritinoctyes
What is the keratinosens assay used to predict?
Skin sensitisation potential of chemicals
