Changes

<p><strong>Flow cytometry</strong> is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous <a title="Parametric model" href="/wiki/Parametric_model" title="Parametric model">multiparametric</a> analysis of the physical and/or <a hrefclass="/wiki/Chemicalmw-redirect" title="Chemical" classhref="mw-redirect/wiki/Chemical">chemical</a> characteristics of single cells flowing through an <a hrefclass="/wiki/Opticalmw-redirect" title="Optical" classhref="mw-redirect/wiki/Optical">optical</a> and/or electronic detection apparatus.</p><table summaryclass="Contentstoc" id="toc" classsummary="tocContents">

<script type="text/javascript">//<![CDATA[ if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } //]]></script><p><a nameid="Principle" idname="Principle"></a></p>

<p>A beam of <a title="Light" href="/wiki/Light" title="Light">light</a> (usually <a title="Laser" href="/wiki/Laser" title="Laser">laser</a> light) of a single wavelength is directed onto a <a title="Hydrodynamic focusing" href="/wiki/Hydrodynamic_focusing" title="Hydrodynamic focusing">hydro-dynamically focused</a> stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more <a hrefclass="/wiki/Fluorescentmw-redirect" title="Fluorescent" classhref="mw-redirect/wiki/Fluorescent">fluorescent</a> detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of <a title="Scattering" href="/wiki/Scattering" title="Scattering">scattered</a> and <a hrefclass="/wiki/Fluorescentmw-redirect" title="Fluorescent" classhref="mw-redirect/wiki/Fluorescent">fluorescent</a> light is picked up by the detectors, and by analysing fluctuations in brightness at each detector (one for each <a hrefclass="/wiki/Emission_spectrum_%28fluorescence_spectroscopy%29mw-redirect" title="Emission spectrum (fluorescence spectroscopy)" classhref="mw-redirect/wiki/Emission_spectrum_%28fluorescence_spectroscopy%29">fluorescent emission peak</a>) it is then possible to extrapolate various types of information about the physical and chemical structure of each individual particle. FSC correlates with the <a title="Cell (biology)" href="/wiki/Cell_%28biology%29" title="Cell (biology)">cell</a> volume and SSC depends on the inner complexity of the particle (i.e. shape of the <a title="Cell nucleus" href="/wiki/Cell_nucleus" title="Cell nucleus">nucleus</a>, the amount and type of <a title="Cytoplasm" href="/wiki/Cytoplasm" title="Cytoplasm">cytoplasmic</a> granules or the <a title="Cell membrane" href="/wiki/Cell_membrane" title="Cell membrane">membrane</a> roughness). Some flow cytometers on the market have eliminated the need for fluorescence and use only light scatter for measurement. Other flow cytometers form images of each cell's fluorescence, scattered light, and transmitted light.</p><p><a nameid="Flow_cytometers" idname="Flow_cytometers"></a></p>

<p>Modern flow cytometers are able to analyse several thousand particles every second, in &quot;real time&quot;, and can actively separate and isolate particles having specified properties. A flow cytometer is similar to a <a title="Microscope" href="/wiki/Microscope" title="Microscope">microscope</a>, except that instead of producing an image of the cell, flow cytometry offers &quot;high-throughput&quot; (for a large number of cells) automated <a title="Quantification" href="/wiki/Quantification" title="Quantification">quantification</a> of set parameters. To analyze solid <a hrefclass="/wiki/Biological_tissuemw-redirect" title="Biological tissue" classhref="mw-redirect/wiki/Biological_tissue">tissues</a> single-cell suspension must first be prepared.</p>

<li>a light source - commonly used are lamps (<a title="Mercury (element)" href="/wiki/Mercury_%28element%29" title="Mercury (element)">mercury</a>, <a title="Xenon" href="/wiki/Xenon" title="Xenon">xenon</a>); high power water-cooled lasers (<a hrefclass="/wiki/Argon_lasermw-redirect" title="Argon laser" classhref="mw-redirect/wiki/Argon_laser">argon</a>, <a hrefclass="/wiki/Krypton_lasermw-redirect" title="Krypton laser" classhref="mw-redirect/wiki/Krypton_laser">krypton</a>, dye laser); low power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); <a hrefclass="/wiki/Diode_lasermw-redirect" title="Diode laser" classhref="mw-redirect/wiki/Diode_laser">diode lasers</a> (blue, green, red, violet). </li>

<li>an amplification system - <a title="Linear" href="/wiki/Linear" title="Linear">linear</a> or <a title="Logarithmic scale" href="/wiki/Logarithmic_scale" title="Logarithmic scale">logarithmic</a>. </li>

<p>Early flow cytometers were generally experimental devices, but recent technological advances have created a considerable market for the instrumentation, as well as the reagents used in analysis, such as <a hrefclass="/wiki/Fluorescent_labelingmw-redirect" title="Fluorescent labeling" classhref="mw-redirect/wiki/Fluorescent_labeling">fluorescently-labeled</a> antibodies and analysis software.</p><p>Modern instruments usually have multiple lasers and fluorescence detectors (the current record for a commercial instrument is 4 lasers and 18 fluorescence detectors). Increasing the number of lasers and detectors allows for multiple antibody labelling, and can more precisely identify a target population by their <a title="Phenotype" href="/wiki/Phenotype" title="Phenotype">phenotype</a>. Certain instruments can even take digital images of individual cells, allowing for the analysis of fluorescent signal location within or on the surface of cells.</p><p>The data generated by flow-cytometers can be plotted in a single <a title="Dimension" href="/wiki/Dimension" title="Dimension">dimension</a>, to produce a <a title="Histogram" href="/wiki/Histogram" title="Histogram">histogram</a>, or in two dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence <a hrefclass="/wiki/Intensitymw-redirect" title="Intensity" classhref="mw-redirect/wiki/Intensity">intensity</a>, by creating a series of subset extractions, termed &quot;gates&quot;. Specific gating protocols exist for diagnostic and clinical purposes especially in relation to <a hrefclass="/wiki/Haematologymw-redirect" title="Haematology" classhref="mw-redirect/wiki/Haematology">haematology</a>. The plots are often made on logarithmic scales. Because different fluorescent dyes' emission spectra overlap <a relclass="nofollowexternal autonumber" hreftitle="http://pingu.salk.edu/flow/fluo.html" titlehref="http://pingu.salk.edu/flow/fluo.html" classrel="external autonumbernofollow">[1]</a>, signals at the detectors have to be compensated electronically as well as computationally.</p><p><a nameid="Fluorescence-activated_cell_sorting" idname="Fluorescence-activated_cell_sorting"></a></p>

<p>Fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological <a hrefclass="/wiki/Cells_%28biology%29mw-redirect" title="Cells (biology)" classhref="mw-redirect/wiki/Cells_%28biology%29">cells</a> into two or more containers, one cell at a time, based upon the specific <a title="Scattering" href="/wiki/Scattering" title="Scattering">light scattering</a> and <a hrefclass="/wiki/Fluorescentmw-redirect" title="Fluorescent" classhref="mw-redirect/wiki/Fluorescent">fluorescent</a> characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The acronym FACS is <a title="Trademark" href="/wiki/Trademark" title="Trademark">trademarked</a> and owned by <a title="Becton Dickinson" href="/wiki/Becton_Dickinson" title="Becton Dickinson">Becton Dickinson</a><sup class="reference" id="_ref-0" class><a title="reference"><a href="#_note-0" title="">[1]</a></sup>. While many immunologists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry. The first cell sorter was invented by Mack Fulwyler in 1965 using the principle of <a class="new" title="Coulter volume (page does not exist)" href="/w/index.php?title=Coulter_volume&amp;action=edit&amp;redlink=1" title="Coulter volume (page does not exist)" class="new">Coulter volume</a> a relatively difficult technique to use for sorting and one no longer used in modern instruments. The technique was expanded by <a title="Leonard Herzenberg" href="/wiki/Leonard_Herzenberg" title="Leonard Herzenberg">Len Herzenberg</a> who was responsible for coining the term FACS. Herzenberg won the <a title="Kyoto Prize" href="/wiki/Kyoto_Prize" title="Kyoto Prize">Kyoto Prize</a> in 2006 for his work in flow cytometry.</p><p>The cell suspension is entrained in the center of a narrow, rapidly flowing stream of <a title="Liquid" href="/wiki/Liquid" title="Liquid">liquid</a>. The flow is arranged so that there is a large separation between cells relative to their <a title="Diameter" href="/wiki/Diameter" title="Diameter">diameter</a>. A <a title="Oscillation" href="/wiki/Oscillation" title="Oscillation">vibrating</a> mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A <a title="Electric charge" href="/wiki/Electric_charge" title="Electric charge">charge</a> is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an <a title="Electrostatic deflection" href="/wiki/Electrostatic_deflection" title="Electrostatic deflection">electrostatic deflection</a> system that diverts droplets into containers based upon their charge. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.</p><p><a nameid="Fluorescent_labels" idname="Fluorescent_labels"></a></p>

<p>The fluorescence labels that can be used, will depend on the lamp or laser used to excite the fluorochromes and on the detectors available:<sup class="reference" id="_ref-1" class><a title="reference"><a href="#_note-1" title="">[2]</a></sup></p><dl><dt>Blue Argon Laser (488 <a class="extiw" title="wiktionary:nanometer" href="http://en.wiktionary.org/wiki/nanometer" title="wiktionary:nanometer" class="extiw">nm</a>) </dt></dl>

<li>Green (usually labelled FL1): <a title="Fluorescein" href="/wiki/Fluorescein" title="Fluorescein">FITC</a>, <a title="Alexa Fluor" href="/wiki/Alexa_Fluor" title="Alexa Fluor">Alexa Fluor&reg; 488</a>, <a title="Green fluorescent protein" href="/wiki/Green_fluorescent_protein" title="Green fluorescent protein">GFP</a>, <a title="CFSE" href="/wiki/CFSE" title="CFSE">CFSE</a>, <a hreftitle="/wiki/CFDA-SE" titlehref="/wiki/CFDA-SE">CFDA-SE</a>, <a title="DyLight Fluor" href="/wiki/DyLight_Fluor" title="DyLight Fluor">DyLight 488</a> </li> <li>Orange (usually FL2): <a title="Phycoerythrin" href="/wiki/Phycoerythrin" title="Phycoerythrin">PE</a> </li> <li>Red channel (usually FL3): <a class="new" title="Peridinin chlorophyll protein (page does not exist)" href="/w/index.php?title=Peridinin_chlorophyll_protein&amp;action=edit&amp;redlink=1" title="Peridinin chlorophyll protein (page does not exist)" class="new">PerCP</a>, <a title="Alexa Fluor" href="/wiki/Alexa_Fluor" title="Alexa Fluor">PE-Alexa Fluor&reg; 700</a>, <a class="new" title="PE-Cy5 (TRI-COLOR®) (page does not exist)" href="/w/index.php?title=PE-Cy5_%28TRI-COLOR%C2%AE%29&amp;action=edit&amp;redlink=1" title="PE-Cy5 (TRI-COLOR®) (page does not exist)" class="new">PE-Cy5 (TRI-COLOR&reg;)</a>, <a class="new" title="PE-Cy5.5 (page does not exist)" href="/w/index.php?title=PE-Cy5.5&amp;action=edit&amp;redlink=1" title="PE-Cy5.5 (page does not exist)" class="new">PE-Cy5.5</a>, <a title="Propidium iodide" href="/wiki/Propidium_iodide" title="Propidium iodide">PI</a> </li> <li>Infra-red (usually FL4; not provided by all FACS machines as standard): <a title="Alexa Fluor" href="/wiki/Alexa_Fluor" title="Alexa Fluor">PE-Alexa Fluor&reg; 750</a>, <a class="new" title="PE-Cy7 (page does not exist)" href="/w/index.php?title=PE-Cy7&amp;action=edit&amp;redlink=1" title="PE-Cy7 (page does not exist)" class="new">PE-Cy7</a> </li>

<li><a title="Allophycocyanin" href="/wiki/Allophycocyanin" title="Allophycocyanin">APC</a> </li>

<p><a nameid="Measurable_parameters" idname="Measurable_parameters"></a></p>

<li>volume and <a title="Morphology (biology)" href="/wiki/Morphology_%28biology%29" title="Morphology (biology)">morphological</a> complexity of cells </li> <li>cell <a title="Pigment" href="/wiki/Pigment" title="Pigment">pigments</a> such as <a title="Chlorophyll" href="/wiki/Chlorophyll" title="Chlorophyll">chlorophyll</a> or <a title="Phycoerythrin" href="/wiki/Phycoerythrin" title="Phycoerythrin">phycoerythrin</a> </li> <li><a title="DNA" href="/wiki/DNA" title="DNA">DNA</a> (<a title="Cell cycle" href="/wiki/Cell_cycle" title="Cell cycle">cell cycle</a> analysis, cell <a title="Kinetic" href="/wiki/Kinetic" title="Kinetic">kinetics</a>, <a title="Proliferation" href="/wiki/Proliferation" title="Proliferation">proliferation</a> etc.) </li> <li><a title="RNA" href="/wiki/RNA" title="RNA">RNA</a> </li> <li><a title="Chromosome" href="/wiki/Chromosome" title="Chromosome">chromosome</a> analysis and sorting (library construction, chromosome paint) </li> <li><a title="Protein" href="/wiki/Protein" title="Protein">protein</a> expression and localization </li> <li>transgenic products <em>in vivo</em>, particularly the <a title="Green fluorescent protein" href="/wiki/Green_fluorescent_protein" title="Green fluorescent protein">Green fluorescent protein</a> or related fluorescent proteins </li> <li>cell surface <a title="Antigen" href="/wiki/Antigen" title="Antigen">antigens</a> (<a title="Cluster of differentiation" href="/wiki/Cluster_of_differentiation" title="Cluster of differentiation">Cluster of differentiation</a> (CD) markers) </li> <li><a title="Intracellular" href="/wiki/Intracellular" title="Intracellular">intracellular</a> antigens (various <a title="Cytokine" href="/wiki/Cytokine" title="Cytokine">cytokines</a>, secondary mediators etc.) </li> <li>nuclear <a title="Antigen" href="/wiki/Antigen" title="Antigen">antigens</a> </li> <li><a title="Enzyme" href="/wiki/Enzyme" title="Enzyme">enzymatic</a> activity </li> <li><a title="PH" href="/wiki/PH" title="PH">pH</a>, intracellular <a hrefclass="/wiki/Ionizemw-redirect" title="Ionize" classhref="mw-redirect/wiki/Ionize">ionized</a> <a title="Calcium" href="/wiki/Calcium" title="Calcium">calcium</a>, <a title="Magnesium" href="/wiki/Magnesium" title="Magnesium">magnesium</a>, <a title="Membrane potential" href="/wiki/Membrane_potential" title="Membrane potential">membrane potential</a> </li> <li>membrane <a hrefclass="/wiki/Fluiditymw-redirect" title="Fluidity" classhref="mw-redirect/wiki/Fluidity">fluidity</a> </li> <li><a title="Apoptosis" href="/wiki/Apoptosis" title="Apoptosis">apoptosis</a> (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, <a title="Caspase" href="/wiki/Caspase" title="Caspase">caspase</a> activity) </li> <li>cell <a title="Viability" href="/wiki/Viability" title="Viability">viability</a> </li>

<li><a hrefclass="/wiki/Oxidative_burstmw-redirect" title="Oxidative burst" classhref="mw-redirect/wiki/Oxidative_burst">oxidative burst</a> </li> <li>characterising <a title="Multidrug resistance" href="/wiki/Multidrug_resistance" title="Multidrug resistance">multidrug resistance</a> (MDR) in cancer cells </li> <li><a title="Glutathione" href="/wiki/Glutathione" title="Glutathione">glutathione</a> </li>

<p><a nameid="Applications" idname="Applications"></a></p>

<p>The technology has applications in a number of fields, including <a title="Molecular biology" href="/wiki/Molecular_biology" title="Molecular biology">molecular biology</a>, <a title="Pathology" href="/wiki/Pathology" title="Pathology">pathology</a>, <a title="Immunology" href="/wiki/Immunology" title="Immunology">immunology</a>, <a hrefclass="/wiki/Plant_biologymw-redirect" title="Plant biology" classhref="mw-redirect/wiki/Plant_biology">plant biology</a> and <a title="Marine biology" href="/wiki/Marine_biology" title="Marine biology">marine biology</a>. In the field of molecular biology it is especially useful when used with fluorescence tagged antibodies. These specific antibodies bind to <a title="Antigen" href="/wiki/Antigen" title="Antigen">antigens</a> on the target cells and help to give information on specific characteristics of the cells being studied in the cytometer. It has broad application in <a title="Medicine" href="/wiki/Medicine" title="Medicine">medicine</a> (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting in IVF). In marine biology, the auto-fluorescent properties of photosynthetic <a title="Plankton" href="/wiki/Plankton" title="Plankton">plankton</a> can be exploited by flow cytometry in order to characterise abundance and community structure. In protein engineering, flow cytometry is used in conjunction with <a title="Yeast display" href="/wiki/Yeast_display" title="Yeast display">yeast display</a> and <a title="Bacterial display" href="/wiki/Bacterial_display" title="Bacterial display">bacterial display</a> to identify cell surface-displayed protein variants with desired properties.</p><p><a nameid="See_also" idname="See_also"></a></p>

<li><a hrefclass="/wiki/Fluorescence_microscopymw-redirect" title="Fluorescence microscopy" classhref="mw-redirect/wiki/Fluorescence_microscopy">Fluorescence microscopy</a> </li>

<p><a nameid="Bibliography" idname="Bibliography"></a></p>

<li>Flow Cytometry First Principles by Alice Longobardi Givan <a class="internal" href="/wiki/Special:Booksources/0471382248" class="internal">ISBN 0471382248</a> </li> <li>Practical Flow Cytometry by Howard M. Shapiro <a class="internal" href="/wiki/Special:Booksources/0471411256" class="internal">ISBN 0471411256</a> </li> <li>Flow Cytometry for Biotechnology by Larry A. Sklar <a class="internal" href="/wiki/Special:Booksources/0195152344" class="internal">ISBN 0195152344</a> </li> <li>Handbook of Flow Cytometry Methods by J. Paul Robinson, et al <a class="internal" href="/wiki/Special:Booksources/0471596345" class="internal">ISBN 0471596345</a> </li>

<li>Ormerod, M.G. (ed.) (2000) Flow cytometry - A practical approach. 3rd edition. Oxford University Press, Oxford, UK. (<a class="internal" href="/wiki/Special:Booksources/0199638241" class="internal">ISBN 0-19-963824-1</a>). </li>

<p><a nameid="References" idname="References"></a></p>

<li id="_note-0"><strong><a title="" href="#_ref-0" title="">^</a></strong> <a relclass="nofollowexternal text" hreftitle="http://www.bdbiosciences.com/pdfs/brochures/23-3428-02.pdf" titlehref="http://www.bdbiosciences.com/pdfs/brochures/23-3428-02.pdf" classrel="external textnofollow">FACS MultiSET System</a> (PDF). Becton Dickinson. Retrieved on <a title="2007" href="/wiki/2007" title="2007">2007</a>-<a title="February 9" href="/wiki/February_9" title="February 9">02-09</a>. </li> <li id="_note-1"><strong><a title="" href="#_ref-1" title="">^</a></strong> <cite style="font-style: normal;">Loken MR (1990). &quot;Immunofluorescence Techniques in Flow Cytometry and Sorting&quot;: 341&ndash;53. Wiley.</cite><span class="Z3988" title="ctx_ver=Z39.88-2004&amp;rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&amp;rft.genre=article&amp;rft.atitle=Immunofluorescence+Techniques+in+Flow+Cytometry+and+Sorting&amp;rft.date=1990&amp;rft.au=Loken+MR&amp;rft.pages=341%26ndash%3B53" class="Z3988"> </span> </li>

<p><a nameid="External_links" idname="External_links"></a></p>

<li><a relclass="nofollowexternal text" hreftitle="http://probes.invitrogen.com/resources/education/" titlehref="http://probes.invitrogen.com/resources/education/" classrel="external textnofollow">Tutorials on fluorescence and flow cytometry</a> </li> <li><a title="Medical Subject Headings" href="/wiki/Medical_Subject_Headings" title="Medical Subject Headings">MeSH</a> <em><a relclass="nofollowexternal text" hreftitle="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&amp;term=Flow+cytometry" titlehref="http://www.nlm.nih.gov/cgi/mesh/2007/MB_cgi?mode=&amp;term=Flow+cytometry" classrel="external textnofollow">Flow+cytometry</a></em> </li> <li><a relclass="nofollowexternal text" hreftitle="http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm" titlehref="http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm" classrel="external textnofollow">Powerpoint lectures on flow cytometry</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html" titlehref="http://sciencepark.mdanderson.org/fcores/flow/files/Operation.html" classrel="external textnofollow">How a flow cytometer operates</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://www.fluorophores.org/" titlehref="http://www.fluorophores.org/" classrel="external textnofollow">Fluorophores.org - The database of fluorescent dyes</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://pingu.salk.edu/flow/fluo.html" titlehref="http://pingu.salk.edu/flow/fluo.html" classrel="external textnofollow">Table of fluorochromes</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://www.bdbiosciences.com/spectra/" titlehref="http://www.bdbiosciences.com/spectra/" classrel="external textnofollow">Java Fluorescence Spectrum Viewer</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://www.cshprotocols.org/cgi/content/full/2007/22/pdb.prot4895" titlehref="http://www.cshprotocols.org/cgi/content/full/2007/22/pdb.prot4895" classrel="external textnofollow">Combined Flow Cytometric Measurement of Two Cell-Surface</a> </li> <li><a relclass="nofollowexternal text" hreftitle="http://www.cytometry.org/" titlehref="http://www.cytometry.org/" classrel="external textnofollow">The Clinical Cytometry Society</a></li>

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