Viability assay
Encyclopedia
A viability assay is an assay
to determine the ability of cell
s or tissue
s 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: (1) the cell membrane
may not be intact
(2) the sodium-potassium pump
may not be operating well As with many kinds of viability assays, quantitative measures of physiological function do not indicate whether damage repair and recovery is possible. Fluorescent
-based assays do not require large sample sizes.
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
to determine the ability of cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s or tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
s to maintain or recover its viability
Viability
Viable or viability is the ability of a thing to maintain itself or recover its potentialities.Viable or viability may also refer to:...
. For example, examining the ratio of potassium
Potassium
Potassium is the chemical element with the symbol K and atomic number 19. Elemental potassium is a soft silvery-white alkali metal that oxidizes rapidly in air and is very reactive with water, generating sufficient heat to ignite the hydrogen emitted in the reaction.Potassium and sodium are...
to sodium
Sodium
Sodium is a chemical element with the symbol Na and atomic number 11. It is a soft, silvery-white, highly reactive metal and is a member of the alkali metals; its only stable isotope is 23Na. It is an abundant element that exists in numerous minerals, most commonly as sodium chloride...
in cells indicates viability because if cells do not have high intracellular potassium and low intracellular sodium: (1) the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
may not be intact
(2) the sodium-potassium pump
Na+/K+-ATPase
Na+/K+-ATPase is an enzyme located in the plasma membrane in all animals.- Sodium-potassium pumps :Active transport is responsible for cells containing relatively high...
may not be operating well As with many kinds of viability assays, quantitative measures of physiological function do not indicate whether damage repair and recovery is possible. Fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
-based assays do not require large sample sizes.
Classification of viability assays
- Cytolysis or membrane leakage assays: This category includes the lactate dehydrogenaseLactate dehydrogenaseLactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
assay, a stable enzyme common in all cells which can be readily detected when cell membranes are no longer intact. Trypan BlueTrypan blueTrypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed;...
assay is also in this category of assay. - Mitochondrial activity or caspase assays: Resazurin and FormazanFormazanFormazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
(MTTMTT assayThe MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes to formazan dyes, giving a purple color. A main application allows to assess the viability and the proliferation of cells...
/XTT) can assay for various stages in the apoptosisApoptosisApoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
process that foreshadow cell death. - Functional assays: Assays of cell function will be highly specific to the types of cells being assayed. For example, motility is a widely-used assay of sperm cell function. FertilityFertilityFertility is the natural capability of producing offsprings. As a measure, "fertility rate" is the number of children born per couple, person or population. Fertility differs from fecundity, which is defined as the potential for reproduction...
can be used to assay gameteGameteA gamete is a cell that fuses with another cell during fertilization in organisms that reproduce sexually...
survival, in general. Red blood cellRed blood cellRed blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
s have been assayed in terms of deformabilityErythrocyte deformabilityErythrocyte deformability refers to the cellular properties of erythrocytes which determine the degree of shape change under a given level of applied force. Erythrocytes change their shape extensively under the influence of applied forces in fluid flow or while passing through microcirculation...
, osmotic fragilityOsmotic fragilityIn hematology, osmotic fragility refers to the degree of hemolysis when red blood cells are placed in a hypotonic solution. In general, osmotic fragility is inversely proportional to the cell's surface-area-to-volume....
, hemolysisHemolysisHemolysis —from the Greek meaning "blood" and meaning a "loosing", "setting free" or "releasing"—is the rupturing of erythrocytes and the release of their contents into surrounding fluid...
, ATPAdenosine triphosphateAdenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
level, and hemoglobinHemoglobinHemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
content. - Genomic and protoemic assays: Cells can be assayed for activation of stress pathways using DNA microarrayDNA microarrayA DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
s and protein chips.
List of common viability assays
- ATP testATP testThe ATP test is a process of rapidly measuring actively growing microorganisms through detection of adenosine triphosphate, or ATP.-ATP testing method:...
- Calcein AM
- Clonogenic assayClonogenic assayA clonogenic assay is a microbiology technique for studying the effectiveness of specific agents on the survival and proliferation of cells. It is frequently used in cancer research laboratories to determine the effect of drugs or radiation on proliferating tumor cells as well as for titration of...
- Ethidium homodimer assayEthidium homodimer assayAn ethidium homodimer assay can be used to detect dead or dying cells. Ethidium homodimer is a membrane-impermeable fluorescent dye which binds to DNA. After a cell sample has been stained with ethidium homodimer, the dead cells may be viewed and counted under a UV-light microscope.When cells die,...
- Evans blueEvans blueT-1824 or Evans Blue, often incorrectly rendered as Evan's Blue, is an azo dye which has a very high affinity for serum albumin. Because of this, it can be useful in physiology in estimating the proportion of body water contained in blood plasma....
- Fluorescein diacetate hydrolysisFluorescein diacetate hydrolysisFluorescein 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...
/Propidium iodidePropidium iodidePropidium 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...
staining (FDA/PI staining) - Flow cytometryFlow cytometryFlow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
- FormazanFormazanFormazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.Leading examples of...
-based assays (MTTMTT assayThe MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes to formazan dyes, giving a purple color. A main application allows to assess the viability and the proliferation of cells...
/XTT) - Green fluorescent proteinGreen fluorescent proteinThe green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
- Lactate dehydrogenaseLactate dehydrogenaseLactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
(LDH) - Methyl violet
- Propidium iodidePropidium iodidePropidium 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...
, DNA stain that can differentiate necroticNecrosisNecrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...
, apoptoticApoptosisApoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
and normal cellsCell (biology)The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
. - Resazurin
- Trypan BlueTrypan blueTrypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed;...
, a living-cell exclusion dye (dye only crosses cell membranes of dead cells) - TUNEL assayTUNEL assayTerminal deoxynucleotidyl transferase dUTP nick end labeling is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.- Method :...