Fluorescein diacetate hydrolysis
Encyclopedia
Fluorescein diacetate hydrolysis assays can be used to measure enzyme activity produced by microbes in a sample. A bright yellow glow is produced and is strongest when enzymatic activity is greatest. This can be quantified using a spectrofluorometer
.
It is often used to measure activity in soil and compost samples, however may not give an accurate reading if microbes with lower activity phases such as esterases cleave the fluorescein
first.
It is also used in combination with propidium iodide
(PI) to determine viability
in eukaryotic cells. Living cells will actively convert the non-fluorescent FDA into the green fluorescent compound "fluorescin", a sign of viability; while nucleus of membrane-compromised cells will fluoresce red, a sign of cell death. Currently FDA/PI staining is the standard assessment of human pancreatic islet viability with suitability for transplantation when viability score is above 70%.
FDA stock solution is prepared by dissolving 5 mg of fluorescein diacetate in 1 ml acetone
, and sucrose
may be added for live cell viability testing. FDA stain must be kept in the dark at 4°C or it will spoil.
Spectrofluorometer
The spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample...
.
It is often used to measure activity in soil and compost samples, however may not give an accurate reading if microbes with lower activity phases such as esterases cleave the fluorescein
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
first.
It is also used in combination with propidium iodide
Propidium iodide
Propidium iodide is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain cells. When excited with 488 nm wavelength light, it fluoresces red...
(PI) to determine viability
Viability assay
A viability assay is an assay to determine the ability of cells or tissues to maintain or recover its viability. For example, examining the ratio of potassium to sodium in cells indicates viability because if cells do not have high intracellular potassium and low intracellular sodium: the...
in eukaryotic cells. Living cells will actively convert the non-fluorescent FDA into the green fluorescent compound "fluorescin", a sign of viability; while nucleus of membrane-compromised cells will fluoresce red, a sign of cell death. Currently FDA/PI staining is the standard assessment of human pancreatic islet viability with suitability for transplantation when viability score is above 70%.
FDA stock solution is prepared by dissolving 5 mg of fluorescein diacetate in 1 ml acetone
Acetone
Acetone is the organic compound with the formula 2CO, a colorless, mobile, flammable liquid, the simplest example of the ketones.Acetone is miscible with water and serves as an important solvent in its own right, typically as the solvent of choice for cleaning purposes in the laboratory...
, and sucrose
Sucrose
Sucrose is the organic compound commonly known as table sugar and sometimes called saccharose. A white, odorless, crystalline powder with a sweet taste, it is best known for its role in human nutrition. The molecule is a disaccharide composed of glucose and fructose with the molecular formula...
may be added for live cell viability testing. FDA stain must be kept in the dark at 4°C or it will spoil.