18 Questions
What is the significance of cellular ATP in measuring cell viability?
It is one of the most sensitive endpoints in measuring cell viability
What is the primary function of luciferase in the ATP assay?
To catalyze the reaction of luciferin to oxyluciferin in the presence of Mg ions and ATP
What is the relationship between the intensity of the luminescent signal and ATP concentration or cell number?
There is a linear relationship
What is the advantage of the ATP assay over other cell viability assays?
It is the fastest, most sensitive, and less prone to artifacts
What is the minimum number of cells that can be detected per well using the ATP assay?
Less than 10 cells
How long does it take for the luminescent signal to reach steady state and stabilize after addition of reagent?
10 minutes
What is an advantage of the Protease Viability Marker Assay (GF-AFC assay) over tetrazolium assays?
Shorter incubation time
What is a potential drawback of the Protease Viability Marker Assay (GF-AFC assay)?
Fluorescent interference from test compounds is possible
What is a characteristic of the signal produced in luminometric assays?
It is a persistent and stable glow-type signal
What is the primary advantage of fluorometric assays over traditional dye exclusion and colorimetric assays?
They are more sensitive
What is an advantage of luminometric assays?
They provide fast and simple determination of cell proliferation and cytotoxicity
What is the function of the GF-AFC substrate in the Protease Viability Marker Assay?
To selectively detect protease activity in viable cells
What is a feature of the Protease Viability Marker Assay (GF-AFC assay)?
It correlates well with other established methods of determining cell viability
What is a characteristic of the Protease Viability Marker Assay (GF-AFC assay)?
It is suitable for multiplexing with other assays
What happens to the protease activity when cells die?
It is rapidly lost
What is the purpose of the aminofluorocoumarin (AFC) in the Protease Viability Marker Assay?
To produce a fluorescent signal proportional to the number of viable cells
What is the advantage of using commercial kits of fluorometric assays?
They are easier to use and experimental procedures are available in kit packages
What type of cells can be used with fluorometric assays?
Both adherent and suspended cell lines
Study Notes
- Fluorometric assays of cell viability and cytotoxicity can be performed using a fluorescence microscope, fluorometer, fluorescence microplate reader, or flow cytometer.
- These assays offer several advantages over traditional dye exclusion and colorimetric assays, including ease of use and applicability for adherent or suspended cell lines.
- Fluorometric assays are more sensitive than colorimetric assays, and commercial kits are available from several companies, with experimental procedures typically provided in kit packages.
- The Protease Viability Marker Assay (GF-AFC assay) measures the conserved and constitutive protease enzyme activity of viable cells, using a cell-permeable fluorogenic protease substrate (glycylphenylalanyl-aminofluorocoumarin; GF-AFC).
- In viable cells, cytoplasmic aminopeptidase activity removes glycine and phenylalanine amino acids, releasing aminofluorocoumarin (AFC) and producing a fluorescent signal proportional to the number of viable cells.
- When cells die, this protease activity is rapidly lost, making it a selective marker of the viable cell population.
- The GF-AFC substrate is relatively nontoxic to cells in culture, and long-term exposure results in little change in cell viability.
- This assay is suitable for multiplexing with other assays, as the cell population remains viable and can be used for subsequent assays.
- The incubation time for the GF-AFC assay is shorter (30 min-1 hour) compared to tetrazolium assays (1-4 hours).
- A disadvantage of the GF-AFC assay is that fluorescent interference from test compounds is possible.
- Luminometric assays provide fast and simple determination of cell proliferation and cytotoxicity in mammalian cells, using a convenient 96-well and 384-well microtiter plate format.
- These assays produce a persistent and stable glow-type signal, allowing for both viability and cytotoxicity values to be obtained from the same well.
- Commercial kits of luminometric assays are available, with experimental procedures typically provided in kit packages.
- The ATP assay measures ATP levels, which represent the most important chemical energy reservoir in cells, used for biological synthesis, signaling, transport, and movement processes.
- When cells are lethally damaged and lose membrane integrity, they lose the ability to synthesize ATP, and the ATP level decreases dramatically.
- The ATP assay is based on the reaction of luciferin to oxyluciferin, catalyzed by the enzyme luciferase in the presence of Mg ions and ATP, yielding a luminescent signal.
- There is a linear relationship between the intensity of the luminescent signal and ATP concentration or cell number.
- The ATP assay chemistry can detect fewer than 10 cells per well, making it suitable for 1536-well plate formats.
- Advantages of the ATP assay include its speed, sensitivity, and low artifact propensity, as well as a fast signal stabilization time (within 10 min) and lack of incubation step for conversion of substrate into a colored compound.
This quiz covers the importance of ATP in cellular processes and its role in measuring cell viability. It also discusses commercial kits available for luminometric assays and their experimental procedures.
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