Editing Flow cytometry (section)

== Applications ==
The technology has applications in a number of fields, including [[molecular biology]], [[pathology]], [[immunology]], virology,<ref>{{cite journal | vauthors = Zamora JL, Aguilar HC | title = Flow virometry as a tool to study viruses | journal = Methods | volume = 134–135 | pages = 87–97 | date = February 2018 | pmid = 29258922 | pmc = 5815898 | doi = 10.1016/j.ymeth.2017.12.011 }}</ref> [[plant biology]] and [[marine biology]].<ref>{{cite journal| vauthors = Murphy RW, Lowcock LA, Smith C, Darevsky IS, Orlov N, MacCulloch RD, Upton DE | title = Flow cytometry in biodiversity surveys: methods, utility and constraints|journal=Amphibia-Reptilia|date=1997|volume=18|pages=1–13|doi=10.1163/156853897x00260}}</ref> It has broad application in [[medicine]] especially in transplantation, hematology, tumor immunology and chemotherapy, prenatal diagnosis, genetics and [[sperm sorting]] for [[sex preselection]]. Flow cytometry is widely applied to detect sperm cells abnormality associated with [[DNA fragmentation]]<ref>{{cite journal | vauthors = Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z | title = Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatic cells | journal = Experimental Cell Research | volume = 207 | issue = 1 | pages = 202–5 | date = July 1993 | pmid = 8391465 | doi = 10.1006/excr.1993.1182 }}</ref> in [[male fertility]] assays.<ref>{{cite journal | vauthors = Evenson DP | title = Evaluation of sperm chromatin structure and DNA strand breaks is an important part of clinical male fertility assessment | journal = Translational Andrology and Urology | volume = 6 | issue = Suppl 4 | pages = S495–S500 | date = September 2017 | pmid = 29082168 | pmc = 5643675 | doi = 10.21037/tau.2017.07.20 | doi-access = free }}</ref> Also, it is extensively used in research for the detection of [[DNA damage]],<ref>{{cite journal | vauthors = Tanaka T, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z | title = Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants | journal = Cell Cycle | volume = 5 | issue = 17 | pages = 1940–5 | date = September 2006 | pmid = 16940754 | pmc = 3488278 | doi = 10.4161/cc.5.17.3191 }}</ref><ref>{{cite journal | vauthors = MacPhail SH, Banáth JP, Yu Y, Chu E, Olive PL | title = Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells | journal = Radiation Research | volume = 159 | issue = 6 | pages = 759–67 | date = June 2003 | pmid = 12751958 | doi = 10.1667/rr3003 | bibcode = 2003RadR..159..759M | s2cid = 26093456 }}</ref> caspase cleavage and [[apoptosis]].<ref>{{cite journal | vauthors = Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F | title = Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis) | journal = Cytometry | volume = 27 | issue = 1 | pages = 1–20 | date = January 1997 | pmid = 9000580 | doi = 10.1002/(SICI)1097-0320(19970101)27:1<1::AID-CYTO2>3.0.CO;2-L | doi-access = free }}</ref> [[Photoacoustic flow cytometry]] is used in the study of multi-drug-resistant bacteria (most commonly MRSA) to detect, differentiate, and quantify bacteria in the blood marked with dyed bacteriophages.<ref>{{Cite book| vauthors = Edgar RH, Noel C, Minard A, Fernandez R, Fitzpatrick M, Sajewski A, Cook J, Hempel JD, Kellum JA, Viator JA | title = Photons Plus Ultrasound: Imaging and Sensing 2019 | chapter = Identification of MRSA infection in blood using photoacoustic flow cytometry | display-authors = 6 |s2cid=86428267 | veditors = Wang L, Oraevsky AA |date=2019-02-27|publisher=International Society for Optics and Photonics|volume=10878|pages=1087860|doi=10.1117/12.2510210|isbn=9781510623989|bibcode=2019SPIE10878E..60E}}</ref> In [[neuroscience]], co-expression of cell surface and intracellular antigens can also be analyzed.<ref>{{cite journal | vauthors = Menon V, Thomas R, Ghale AR, Reinhard C, Pruszak J | title = Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types | journal = Journal of Visualized Experiments | issue = 94 | pages = e52241 | date = December 2014 | pmid = 25549236 | pmc = 4396953 | doi = 10.3791/52241 }}</ref> In microbiology, it can be used to screen and sort transposon mutant libraries constructed with a GFP-encoding transposon (TnMHA),<ref>{{cite journal | vauthors = Antypas H, Veses-Garcia M, Weibull E, Andersson-Svahn H, Richter-Dahlfors A | title = A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide | journal = Lab on a Chip | volume = 18 | issue = 12 | pages = 1767–1777 | date = June 2018 | pmid = 29781496 | pmc = 5996734 | doi = 10.1039/c8lc00190a }}</ref> or to assess viability.<ref name="pmid21705550">{{cite journal | vauthors = Davey HM | title = Life, death, and in-between: meanings and methods in microbiology | journal = Applied and Environmental Microbiology | volume = 77 | issue = 16 | pages = 5571–6 | date = August 2011 | pmid = 21705550 | pmc = 3165249 | doi = 10.1128/AEM.00744-11 | bibcode = 2011ApEnM..77.5571D }}</ref> In protein engineering, flow cytometry is used in conjunction with [[yeast display]] and [[bacterial display]] to identify cell surface-displayed protein variants with desired properties.
The main advantages of flow cytometry over histology and IHC is the possibility to precisely measure the quantities of antigens and the possibility to stain each cell with multiple antibodies-fluorophores, in current laboratories around 10 antibodies can be bound to each cell. This is much less than mass cytometer where up to 40 can be currently measured, but at a higher price and a slower pace.

=== Aquatic research ===
In aquatic systems, flow cytometry is used for the analysis of autofluorescing cells or cells that are fluorescently-labeled with added stains. This research started in 1981 when [[Clarice Yentsch]] used flow cytometry to measure the fluorescence in a red tide producing dinoflagellate.<ref>{{cite journal | vauthors = Yentsch CM | title = Flow cytometric analysis of cellular saxitoxin in the dinoflagellate Gonyaulax tamarensis var. excavata | journal = Toxicon | volume = 19 | issue = 5 | pages = 611–21 | date = 1981 | pmid = 7197816 | doi = 10.1016/0041-0101(81)90099-4 | bibcode = 1981Txcn...19..611Y }}</ref> The next year researchers published flow cytometric measurements of multiple algal species which could be distinguished based on their fluorescence characteristics.<ref>{{Cite journal|last1=Trask|first1=B. J.|last2=Engh|first2=G. J. van den|last3=Elgershuizen|first3=J. H. B. W.|date=1982|title=Analysis of phytoplankton by flow cytometry|journal=Cytometry|language=en|volume=2|issue=4|pages=258–264|doi=10.1002/cyto.990020410|pmid=6799265|issn=1097-0320|doi-access=free}}</ref> By 1983, marine researchers were assembling their own flow cytometers<ref>{{Cite journal|last1=Olson|first1=Robert J.|last2=Frankel|first2=Sheila L.|last3=Chisholm|first3=Sallie W.|last4=Shapiro|first4=Howard M.|date=1983-04-08|title=An inexpensive flow cytometer for the analysis of fluorescence signals in phytoplankton: Chlorophyll and DNA distributions|url=https://dx.doi.org/10.1016/0022-0981%2883%2990155-7|journal=Journal of Experimental Marine Biology and Ecology|language=en|volume=68|issue=2|pages=129–144|doi=10.1016/0022-0981(83)90155-7|bibcode=1983JEMBE..68..129O |issn=0022-0981|url-access=subscription}}</ref> or using commercially available flow cytometers on seawater samples collected off Bermuda to demonstrate that phytoplankton cells could be distinguished from non-living material and that cyanobacteria could be sorted from a mixed community and subsequently cultured in the lab.<ref>{{Cite journal| vauthors = Yentsch CM, Horan PK, Muirhead K, Dortch Q, Haugen E, Legendre L, Murphy LS, Perry MJ, Phinney DA, Pomponi SA, Spinrad RW | display-authors = 6 |date=1983|title=Flow cytometry and cell sorting: A technique for analysis and sorting of aquatic particles1 |journal=Limnology and Oceanography|language=en|volume=28|issue=6|pages=1275–1280|doi=10.4319/lo.1983.28.6.1275| bibcode = 1983LimOc..28.1275Y |issn=1939-5590|doi-access=free}}</ref> Flow cytometry also allowed marine researchers to distinguish between dimly-fluorescing ''[[Prochlorococcus]]'' and heterotrophic microorganisms, a distinction that is difficult with microscopy-based assessments.<ref>{{Cite journal| vauthors = Chisholm SW, Olson RJ, Zettler ER, Goericke R, Waterbury JB, Welschmeyer NA |date= July 1988 |title=A novel free-living prochlorophyte abundant in the oceanic euphotic zone |journal=Nature |volume=334|issue=6180|pages=340–343|doi=10.1038/334340a0 |bibcode= 1988Natur.334..340C |s2cid= 4373102 }}</ref> Advances in technology now allow aquatic scientists to use flow cytometers continuously during research cruises<ref>{{Cite journal| vauthors = Swalwell JE, Ribalet F, Armbrust EV |date=2011|title=SeaFlow: A novel underway flow-cytometer for continuous observations of phytoplankton in the ocean |journal=Limnology and Oceanography: Methods|language=en|volume=9|issue=10|pages=466–477|doi=10.4319/lom.2011.9.466|issn=1541-5856|doi-access=free|bibcode=2011LimOM...9..466S }}</ref> and flow cytometers are used to provide images of individual phytoplankton cells.<ref>{{Cite journal| vauthors = Olson RJ, Sosik HM |date=2007|title=A submersible imaging-in-flow instrument to analyze nano-and microplankton: Imaging FlowCytobot |journal=Limnology and Oceanography: Methods|language=en|volume=5|issue=6|pages=195–203|doi=10.4319/lom.2007.5.195 |doi-access=free|bibcode=2007LimOM...5..195O }}</ref><ref>{{Cite journal| vauthors = Jakobsen HH, Carstensen J |date=2011|title=FlowCAM: Sizing cells and understanding the impact of size distributions on biovolume of planktonic community structure |journal=Aquatic Microbial Ecology|language=en|volume=65|issue=1|pages=75–87|doi=10.3354/ame01539|issn=0948-3055|doi-access=free}}</ref> Marine scientists use the sorting ability of flow cytometers to make discrete measurements of cellular activity and diversity,<ref>{{cite journal | vauthors = Longnecker K, Sherr BF, Sherr EB | title = Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem | journal = Applied and Environmental Microbiology | volume = 71 | issue = 12 | pages = 7737–49 | date = December 2005 | pmid = 16332746 | pmc = 1317353 | doi = 10.1128/AEM.71.12.7737-7749.2005 | bibcode = 2005ApEnM..71.7737L }}</ref><ref>{{cite journal | vauthors = Stepanauskas R, Sieracki ME | title = Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 21 | pages = 9052–7 | date = May 2007 | pmid = 17502618 | pmc = 1885626 | doi = 10.1073/pnas.0700496104 | bibcode = 2007PNAS..104.9052S | doi-access = free }}</ref> to conduct investigations into the mutualistic relationships between microorganisms that live in close proximity,<ref>{{cite journal | vauthors = Thompson AW, Foster RA, Krupke A, Carter BJ, Musat N, Vaulot D, Kuypers MM, Zehr JP | display-authors = 6 | title = Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga | journal = Science | volume = 337 | issue = 6101 | pages = 1546–50 | date = September 2012 | pmid = 22997339 | doi = 10.1126/science.1222700 | bibcode = 2012Sci...337.1546T | s2cid = 7071725 }}</ref> and to measure biogeochemical rates of multiple processes in the ocean.<ref>{{Cite journal|last1=Lomas|first1=Michael W.|last2=Bronk|first2=Deborah A.|last3=van den Engh|first3=Ger|date=2011-01-15|title=Use of Flow Cytometry to Measure Biogeochemical Rates and Processes in the Ocean|url=http://www.annualreviews.org/doi/10.1146/annurev-marine-120709-142834|journal=Annual Review of Marine Science|language=en|volume=3|issue=1|pages=537–566|doi=10.1146/annurev-marine-120709-142834|pmid=21329216|bibcode=2011ARMS....3..537L|issn=1941-1405|url-access=subscription}}</ref>

=== Cell proliferation assay ===
Cell proliferation is the major function in the immune system. Often it is required to analyse the proliferative nature of the cells in order to make some conclusions. One such assay to determine the cell proliferation is the tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor proliferative cells. This assay gives quantitative as well as qualitative data during time-series experiments.<ref name="pmid17853861">{{cite journal | vauthors = Hawkins ED, Hommel M, Turner ML, Battye FL, Markham JF, Hodgkin PD | title = Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data | journal = Nature Protocols | volume = 2 | issue = 9 | pages = 2057–67 | year = 2007 | pmid = 17853861 | doi = 10.1038/nprot.2007.297 | s2cid = 13550456 }}</ref> This dye binds covalently with the long-lived molecules present inside the cell. When the cells divide, the molecules divide too and, the daughter cells possess half the dye than the parent population. This decrease in the intensity can be visualized by flow cytometry.<ref name="pmid20972413">{{cite journal | vauthors = Quah BJ, Parish CR | title = The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation | journal = Journal of Visualized Experiments | issue = 44 | date = October 2010 | pmid = 20972413 | pmc = 3185625 | doi = 10.3791/2259 }}</ref> In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines.<ref>{{cite journal | vauthors = Jedema I, van der Werff NM, Barge RM, Willemze R, Falkenburg JH | title = New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population | journal = Blood | volume = 103 | issue = 7 | pages = 2677–82 | date = April 2004 | pmid = 14630824 | doi = 10.1182/blood-2003-06-2070 | s2cid = 1984056 | doi-access = free }}</ref>

=== Measuring genome size ===
Flow cytometry has been used to measure [[genome size]]s, or more precisely: the amount of [[DNA]] in a [[Cell (biology)|cell]] or [[Cell nucleus|nucleus]]. Although genomes can be analyzed with more precision by [[Whole genome sequencing|genome sequencing]], this is often difficult due to a high fraction of [[Microchromosome|micro-chromosomes]] or [[Repeated sequence (DNA)|repetitive sequences]] which may be missed by sequencing (or which get filtered out during the analysis step when they cannot be assigned to [[chromosome]]s). However, flow cytometry is not perfect either. The resulting genome sizes may differ based on the dye used. An analysis of fish genomes resulted in significantly different genome sizes when [[propidium iodide]] (PI) and [[DAPI]] were used, respectively. For instance, the genome of ''[[Japanese eel|Anguilla japonica]]'' was found to contain 1.09 pg of DNA with PI vs. 1.25 pg with DAPI. Similarly, the genome of ''[[Chinese high-fin banded shark|Myxocyprinus asiaticus]]'' was found to contain 2.75 pg of DNA (PI) vs. 3.08 pg (DAPI). That is, the differences were on the order of 12–14%.<ref>{{Cite journal |last1=Zhu |first1=Dongmei |last2=Song |first2=Wen |last3=Yang |first3=Kun |last4=Cao |first4=Xiaojuan |last5=Gul |first5=Yasmeen |last6=Wang |first6=Weiming |date=2012 |title=Flow cytometric determination of genome size for eight commercially important fish species in China |url=https://www.jstor.org/stable/23279365 |journal=In Vitro Cellular & Developmental Biology. Animal |volume=48 |issue=8 |pages=507–517 |doi=10.1007/s11626-012-9543-7 |jstor=23279365 |pmid=22956044 |s2cid=255351169 |issn=1071-2690|url-access=subscription }}</ref>