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Physical fractionation is a process by which continuously dividing cells are separated into phase-enriched populations based on characteristics such as the following:
*Cell density.
*Cell size
*The presence of cell surface [[epitope]]s marked by [[antibodies]]
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===[[Counterflow centrifugation elutriation|Centrifugal Elutriation]] ===
'''(Previously called: counter streaming centrifugation)''' Centrifugal [[elutriation]] can be used to separate cells in different phases of the cell cycle based on their size and sedimentation velocity (related to [[sedimentation coefficient]]). Because of the consistent growth patterns throughout the cell cycle, centrifugal elutriation can separate cells into [[G1 phase|G1]], [[S phase|S]], [[G2 phase|G2]], and [[M-phase|M]] phases by increasing size (and increasing sedimentation coefficients) with diminished resolution between G2 and M phases due to cellular heterogeneity and lack of a distinct size change.<ref>{{Cite journal|last=Keng|first=PC|date=September 1980|title=Synchronization of 9L rat brain tumor cells by centrifugal elutriation.|journal=Cell Biophysics|volume=2|issue=3|pages=191–206|pmid=6159093|doi=10.1007/BF02790449|s2cid=7679022}}</ref>
Larger cells sediment faster, so a cell in G2, which has experienced more growth time, will sediment faster than a cell in G1 and can therefore be fractionated out. Cells grown in suspension tend to be easier to elutriate given that they do not adhere to one another and have rounded, uniform shapes. However, some types of adherent cells can be treated with [[trypsin]] and resuspended for elutriation as they will assume a more rounded shape in suspension.<ref>{{Cite book|title=Methods in Enzymology|last=Krek|first=Wilhelm|publisher=Elsevier Inc.|year=1995
===[[Flow cytometry|Flow Cytometry]] and [[Cell sorting|Cell Sorting]]===
[[Flow cytometry]] allows for detection, counting, and measurement of the physical and chemical properties of cells. Cells are suspended in fluid and put through the flow cytometer. Cells are sent one at a time through a laser beam and the light scatter is measured by a detector. Cells or their components can be labeled with fluorescent markers so that they emit different wavelengths of light in response to the laser, allowing for additional data collection.
For quantitative cell cycle analysis, cells are usually fixed with ethanol and stained with DNA-binding dyes like [[propidium iodide]], [[Bisbenzimide|Hoechst 33342]], [[DAPI]], [[7-Aminoactinomycin D|7-Aminoactinomysin D]], [[Plicamycin|Mithramycin]], [[Anthraquinone|DRAQ5]], or TO-PRO-3, allowing for determination of phase by DNA quantity.<ref name=":0">Banfalvi G. (2011) Overview of Cell Synchronization. In: Banfalvi G. (eds) Cell Cycle Synchronization. Methods in Molecular Biology (Methods and Protocols), vol 761. Humana Press.</ref> However, if these cells have been fixed, they are dead and cannot be maintained for continued growth. Cells can also be resuspended in media and dyed with non-toxic dyes to maintain living cultures. Cells can also be [[Genome editing|genome edited]] such that some cellular proteins are made with conjugated fluorescent tags such as [[Green fluorescent protein|GFP]], [[mCherry]], and [[Luciferase]] that can be used to detect and quantify those components. For example, chimeric [[histone H2B]]-GFP constructs can be made and used to measure DNA content and determine replication status as a means of discerning cell phase.<ref>{{Cite journal|last=Coquelle|first=A|
Flow cytometers can be used to collect multiparameter cytometry data, but cannot be used to separate or purify cells. [[Fluorescence-activated cell sorting]] (FACS) is a technique for sorting out the cells based on the differences that can be detected by light scatter (e.g. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins or antigens). The system works much like flow cytometry, but will also charge each cell droplet after it has been measured based on a defined parameter. The charged droplet will then encounter an [[electrostatic deflection]] system that will sort the cell to a different container based on that charge. This allows cells to be separated on the basis of fluorescent content or scatter.
To summarize, flow cytometry alone can be used to gather quantitative data about cell cycle phase distribution, but flow cytometry in coordination with FACS can be used to gather quantitative data and separate cells by phase for further study. Limitations include:
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==Chemical blockade==
The addition of [[exogenous]] substrates can be used to block cells in certain phases of the cell cycle and frequently target [[
* the proportion of synchronized cells is insufficient
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==== [[Nocodazole]] ====
Nocodazole is a rapidly-reversible inhibitor of [[microtubule]] [[polymerization]] that can be used to arrest cells before [[Anaphase]] at the [[Spindle checkpoint|spindle assembly checkpoint]] in the metaphase/anaphase transition. The microtubule poison works by blocking the formation of the [[Spindle apparatus|mitotic spindles]] that attach to and pull apart [[sister chromatids]] in dividing cells. Cells will remain arrested until the nocodazole has been washed out. Nocodazole does not appear to disrupt interphase metabolism, and released cells return to normal cell cycle progression.<ref>{{Cite journal|
==== Inhibition of [[Cyclin-dependent kinase 1|CDK1]] ====
CDK1 is necessary for the transition from G2 to M phase. RO-3306 is a selective CDK1 inhibitor that can reversibly arrest cells at the G2/M border. RO-3306 synchronized >95% of cycling cells (including cancer cells), and released cells rapidly enter mitosis.<ref>{{Cite journal|last=Vassilev|first=Lyubomir T|date=8 November 2006|title=Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1
==== [[Roscovitine]] ====
Roscovitine can be used to inhibit the activity of [[
==== [[Colchicine]] ====
=== Arrest in S (G1/S arrest) ===
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==== [[Hydroxycarbamide|Hydroxyurea]] ====
Hydroxyurea decreases the production of [[Dideoxynucleotide|dNTPs]] by inhibiting the enzyme [[ribonucleotide reductase]]. This serves to halt DNA synthesis by depriving [[DNA polymerase]] of dNTPs at [[
==== [[Aphidicolin]] ====
Aphidocolin is a fungus-derived tetracyclic [[diterpenoid]] that acts as a selective inhibitor for [[DNA polymerase alpha|DNA polymerase α]].
=== Arrest in G1 ===
A single commonly
== Other Methods of Synchronization ==
=== Mitotic Selection ===
Mitotic selection is a drug-free procedure for the selection of mitotic cells from a monolayer undergoing exponential growth.<ref>{{Cite web|url=https://www.biology-online.org/dictionary/Mitotic_cell_selection|title=Mitotic cell selection
===Nutrient/Serum Deprivation===
Elimination of [[Blood plasma|serum]] from the [[culture medium]] for about 24 hours results in the accumulation of cells at the transition between [[G0 phase|G<sub>0</sub>]] quiescence and early G1. This arrest is easily reversible through addition of serum or the deprived nutrient. Upon release, progression through the cell cycle is variable, as some cells remain quiescent while others proceed through the cell cycle at variable rates.<ref name=":1">{{Cite journal|
=== Contact Inhibition ===
Contact inhibition occurs when cells are allowed to grow to high or full confluence, maximizing cell-to-cell contact. This triggers arrest in early G1 in normal cells. Arrest is reversed by replating cells at a lower density.<ref name=":1" /> Because of the proliferation-promoting mutations intrinsic to cancer, tumor cell lines are not usually able to undergo contact inhibition, though there are exceptions.<ref>{{Cite journal|
==External links==
*[https://archive.today/20141014163501/http://www.scienceboard.net/protocol/29/metaphase-block-for-cell-synchronization Cell synchronization protocol]
==References==
{{reflist}}
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