Propidium iodide
Encyclopedia
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. Propidium iodide is used as a DNA stain for both flow cytometry
to evaluate cell viability or DNA content in cell cycle analysis
and microscopy to visualise the nucleus and other DNA containing organelles. It can be used to differentiate necrotic
, apoptotic
and normal cells
.
Propidium Iodide is the most commonly used dye to quantitatively assess DNA content.
Propidium iodide (PI) binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. PI also binds to RNA
, necessitating treatment with nucleases to distinguish between RNA and DNA staining. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, the fluorescence excitation maximum is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to the blue. Although its molar absorptivity (extinction coefficient) is relatively low, PI exhibits a sufficiently large Stokes shift
to allow simultaneous detection of nuclear DNA and fluorescein
-labeled antibodies, provided the proper optical filters are used. PI is suitable for fluorescence microscopy, confocal laser scanning microscopy
, flow cytometry
, and fluorometry.
PI is membrane impermeant and generally excluded from viable cells. PI is commonly used for
identifying dead cells in a population and as a counterstain in multicolor fluorescent techniques. The counterstaining protocols below are compatible with a wide range of cytological labeling techniques—direct or indirect antibody-based detection methods, mRNA in situ hybridization, or staining with fluorescent reagents specific for cellular structures. These protocols can be modified for tissue staining.
A typical use of propidium iodide in plant biology is to stain the cell wall. Especially useful for Arabidopsis thaliana
seedling root tissue observed by confocal microscopy
, it increases visibility of the outlines of cells in the root tip. This red fluorescent background is useful to determine the sub-localization of a gene of interest expressed as a Green Fluorescent Protein
fusion.
Also, propidium iodide is used as a stain in animal cells. For example, in Apodemus sylvaticus, more commonly known as the wood mouse
, it can be used to indicate the location of the nuclear region by emitting its characteristic red fluorescence.
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
with a molecular mass
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
of 668.4 Da
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
that can be used to stain
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...
cells
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....
. When excited with 488 nm wavelength light, it fluoresces red. Propidium iodide is used as a DNA stain for both flow cytometry
Flow cytometry
Flow 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...
to evaluate cell viability or DNA content in cell cycle analysis
Cell cycle analysis
Cell cycle analysis is a method in cell biology that employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are permeabilised and treated with a fluorescent dye that stains DNA quantitatively, usually propidium iodide...
and microscopy to visualise the nucleus and other DNA containing organelles. It can be used to differentiate necrotic
Necrosis
Necrosis 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...
, apoptotic
Apoptosis
Apoptosis 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 cells
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....
.
Propidium Iodide is the most commonly used dye to quantitatively assess DNA content.
Propidium iodide (PI) binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. PI also binds to RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, necessitating treatment with nucleases to distinguish between RNA and DNA staining. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold, the fluorescence excitation maximum is shifted ~30–40 nm to the red and the fluorescence emission maximum is shifted ~15 nm to the blue. Although its molar absorptivity (extinction coefficient) is relatively low, PI exhibits a sufficiently large Stokes shift
Stokes shift
Stokes shift is the difference between positions of the band maxima of the absorption and emission spectra of the same electronic transition. It is named after Irish physicist George G. Stokes. When a system absorbs a photon, it gains energy and enters an excited state...
to allow simultaneous detection of nuclear DNA and 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....
-labeled antibodies, provided the proper optical filters are used. PI is suitable for fluorescence microscopy, confocal laser scanning microscopy
Confocal laser scanning microscopy
Confocal laser scanning microscopy is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning...
, flow cytometry
Flow cytometry
Flow 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...
, and fluorometry.
PI is membrane impermeant and generally excluded from viable cells. PI is commonly used for
identifying dead cells in a population and as a counterstain in multicolor fluorescent techniques. The counterstaining protocols below are compatible with a wide range of cytological labeling techniques—direct or indirect antibody-based detection methods, mRNA in situ hybridization, or staining with fluorescent reagents specific for cellular structures. These protocols can be modified for tissue staining.
A typical use of propidium iodide in plant biology is to stain the cell wall. Especially useful for Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
seedling root tissue observed by confocal microscopy
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
, it increases visibility of the outlines of cells in the root tip. This red fluorescent background is useful to determine the sub-localization of a gene of interest expressed as a Green Fluorescent Protein
Green fluorescent protein
The 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...
fusion.
Also, propidium iodide is used as a stain in animal cells. For example, in Apodemus sylvaticus, more commonly known as the wood mouse
Wood mouse
The wood mouse is a common murid rodent from Europe and northwestern Africa. It is closely related to the yellow-necked mouse but differs in that it has no band of yellow fur around the neck, has slightly smaller ears, and is usually slightly smaller overall: around 90 mm in length...
, it can be used to indicate the location of the nuclear region by emitting its characteristic red fluorescence.