Cell cycle analysis
Encyclopedia
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
(PI). The fluorescence intensity of the stained cells at certain wavelengths will therefore correlate with the amount of DNA they contain. As the DNA content of cells duplicates during the S phase
of the cell cycle, the relative amount of cells in the G0 phase
and G1 phase
(before S phase), in the S phase, and in the G2 phase
and M phase (after S phase) can be determined, as the fluorescence of cells in the G2/M phase will be twice as high as that of cells in the G0/G1 phase.
Cell cycle anomalies can be symptoms for various kinds of cell damage, for example DNA damage, which cause the cell to interrupt the cell cycle at certain checkpoints
to prevent transformation into a cancer cell (carcinogenesis
). Other possible reasons for anomalies include lack of nutrients, for example after serum
deprivation.
Cell cycle analysis was first described in 1969 at Los Alamos Scientific Laboratory by a group from the University of California
, using the Feulgen stain
ing technique. The first protocol for cell cycle analysis using propidium iodide staining was presented in 1975 by Awtar Krishan from Harvard Medical School
and is still widely cited today.
The first step in preparing cells for cell cycle analysis is permeabilisation of the cells' plasma membranes. This is usually done by incubating them in a buffer solution
containing a detergent
such as Triton X-100 or NP-40
, or by fixating
them in ethanol
. Most fluorescent DNA dyes are not membrane permeable, that is, not able to pass through an intact cell membrane. Permeabilisation is therefore crucial for the success of the next step, the staining of the cells.
Prior to or during the staining step, the cells will usually be treated with RNase A to remove RNA
s from the cells. This is important because dyes that stain DNA will also stain RNA, thus creating artefact
s that would distort the results.
Aside from propidium iodide, dyes that are frequently used include (but are not limited to) 7-Aminoactinomycin D, DAPI
and Hoechst 33342. All of them have in common that they intercalate
in DNA molecules (see image), thus staining it quantitatively.
When the cells pass through the flow cytometer's laser
, a fluorescence pulse is generated that correlates with the amount of dye intercalated in the DNA and thus with the total amount of DNA in the cell.
. During apoptosis
, the process of programmed cell death, DNA is degraded by cellular endonuclease
s. Therefore, Nuclei
of apoptotic cells contain less DNA than nuclei of healthy G0/G1 cells, resulting in a sub-G0/G1 peak in the fluorescence histogram
that can be used to quantify the amount of apoptotic cells in a sample.
This method was first described in 1991 by a group from Perugia University
School of Medicine. An optimised protocol developed by two of the authors of the original publication was published in 2006
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
that employs 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 distinguish cells in different phases of the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
. Before analysis, the cells are permeabilised and treated with a fluorescent dye that stains DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
quantitatively, usually 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). The fluorescence intensity of the stained cells at certain wavelengths will therefore correlate with the amount of DNA they contain. As the DNA content of cells duplicates during the S phase
S phase
S-phase is the part of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Precise and accurate DNA replication is necessary to prevent genetic abnormalities which often lead to cell death or disease. Due to the importance, the regulatory pathways that govern this...
of the cell cycle, the relative amount of cells in the G0 phase
G0 phase
The G0 phase is a period in the cell cycle in which cells exist in a quiescent state. G0 phase is viewed as either an extended G1 phase, where the cell is neither dividing nor preparing to divide, or a distinct quiescent stage that occurs outside of the cell cycle...
and G1 phase
G1 phase
The G1 phase is a period in the cell cycle during interphase, before the S phase. For many cells, this phase is the major period of cell growth during its lifespan. During this stage new organelles are being synthesized, so the cell requires both structural proteins and enzymes, resulting in great...
(before S phase), in the S phase, and in the G2 phase
G2 phase
G2 phase is the 3rd and final subphase of Interphase in the cell cycle directly preceding Mitosis. It follows the successful completion of S phase, during which the cell’s DNA is replicated...
and M phase (after S phase) can be determined, as the fluorescence of cells in the G2/M phase will be twice as high as that of cells in the G0/G1 phase.
Cell cycle anomalies can be symptoms for various kinds of cell damage, for example DNA damage, which cause the cell to interrupt the cell cycle at certain checkpoints
Cell cycle checkpoint
Cell cycle checkpoints are control mechanisms that ensure the fidelity of cell division in eukaryotic cells. These checkpoints verify whether the processes at each phase of the cell cycle have been accurately completed before progression into the next phase...
to prevent transformation into a cancer cell (carcinogenesis
Carcinogenesis
Carcinogenesis or oncogenesis is literally the creation of cancer. It is a process by which normal cells are transformed into cancer cells...
). Other possible reasons for anomalies include lack of nutrients, for example after serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
deprivation.
Cell cycle analysis was first described in 1969 at Los Alamos Scientific Laboratory by a group from the University of California
University of California
The University of California is a public university system in the U.S. state of California. Under the California Master Plan for Higher Education, the University of California is a part of the state's three-tier public higher education system, which also includes the California State University...
, using the Feulgen stain
Feulgen stain
Feulgen stain is a staining technique discovered by Robert Feulgen and used in histology to identify chromosomal material or DNA in cell specimens. It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided....
ing technique. The first protocol for cell cycle analysis using propidium iodide staining was presented in 1975 by Awtar Krishan from Harvard Medical School
Harvard Medical School
Harvard Medical School is the graduate medical school of Harvard University. It is located in the Longwood Medical Area of the Mission Hill neighborhood of Boston, Massachusetts....
and is still widely cited today.
Experimental procedure
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
containing a detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
such as Triton X-100 or NP-40
NP-40
NP-40 is a commercially available detergent. The full name of NP-40 is Tergitol-type NP-40, which is nonyl phenoxypolyethoxylethanol.Care should be taken to avoid confusing NP-40 with Nonidet P-40 ....
, or by fixating
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...
them in ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
. Most fluorescent DNA dyes are not membrane permeable, that is, not able to pass through an intact cell membrane. Permeabilisation is therefore crucial for the success of the next step, the staining of the cells.
Prior to or during the staining step, the cells will usually be treated with RNase A to remove RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
s from the cells. This is important because dyes that stain DNA will also stain RNA, thus creating artefact
Artifact (error)
In natural science and signal processing, an artifact is any error in the perception or representation of any visual or aural information introduced by the involved equipment or technique....
s that would distort the results.
Aside from propidium iodide, dyes that are frequently used include (but are not limited to) 7-Aminoactinomycin D, DAPI
DAPI
DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy...
and Hoechst 33342. All of them have in common that they intercalate
Intercalation (chemistry)
In chemistry, intercalation is the reversible inclusion of a molecule between two other molecules . Examples include DNA intercalation and graphite intercalation compounds.- DNA intercalation :...
in DNA molecules (see image), thus staining it quantitatively.
When the cells pass through the flow cytometer's laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...
, a fluorescence pulse is generated that correlates with the amount of dye intercalated in the DNA and thus with the total amount of DNA in the cell.
Doublet discrimination
Because cells and especially fixated cells tend to stick together, cell aggregates have to be excluded from analysis through a process called doublet discrimination. This is important because a doublet of two cells in the G0/G1 phase has the same total amount of DNA and thus the same fluorescence intensity as a single cell in the G2/M phase.. G0/G1 doublets would therefore create false positive results for G2/M cells.Related Methods
Because cell cycle analysis is based on measuring the DNA content of cells, it can also be used to detect apoptotic DNA fragmentationApoptosis DNA Fragmentation
Apoptosis DNA fragmentation is a key feature of programmed cell death and also occurs in certain stages of necrosis. Apoptosis is characterized by the activation of endogenous endonucleases with subsequent cleavage of chromatin DNA into internucleosomal fragments of 180 BP and multiples thereof.DNA...
. During apoptosis
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...
, the process of programmed cell death, DNA is degraded by cellular endonuclease
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
s. Therefore, Nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
of apoptotic cells contain less DNA than nuclei of healthy G0/G1 cells, resulting in a sub-G0/G1 peak in the fluorescence histogram
Histogram
In statistics, a histogram is a graphical representation showing a visual impression of the distribution of data. It is an estimate of the probability distribution of a continuous variable and was first introduced by Karl Pearson...
that can be used to quantify the amount of apoptotic cells in a sample.
This method was first described in 1991 by a group from Perugia University
University of Perugia
University of Perugia is a public-owned university based in Perugia, Italy. It was founded in 1308, as attested by the Bull issued by Pope Clement V certifying the birth of the Studium Generale....
School of Medicine. An optimised protocol developed by two of the authors of the original publication was published in 2006