Jump to content

User:ApexofAdharma/sandbox

From Wikipedia, the free encyclopedia

Fluorescent activated[edit]

[edit]

Main article: Flow cytometry

Fluorescent Activated Cell Sorting, or FACS, utilizes Flow cytometry to provide a fast, objective and quantitative measurement of intra- and extracellular properties, not including morphology, for sorting a heterogeneous mixture of cells. This is the most common method of currently separating cells and involves encapsulating cells into small liquid droplets and selectively labeled with electric charges and sorted by an external electric field. FACS has several systems that work together to achieve successful sorting of events of interest. These include fluidics, optics, sorting systems. Utilizing fluorescence, users can identify a particular population of events within a sample containing many differently characterized cells. This technology is used extensively in hematology labs. However, researchers can use a variety of fluorescent dyes to design multi-color panels to achieve successful, simultaneous sorting of multiple, precisely defined cell-types.

Fluorescent Dyes in Cell Sorting

[edit]
A diagram of the sorting system in FACS. After passing through the analysis point and receiving a variable charge, a sample stream passes through an electric field generated by two voltage plates. Depending on the parameters of the experiment, the charged droplets will be deflected into collection tubes. Unwanted events fall straight down into a waste trough.

Fluorescent dyes can act very differently. Generally, a fluorescent dye will be excited by a coherent light source (a laser) at a particular wavelength and emit light at a lower energy and longer wavelength. The most common dyes act by binding to antigens presented on cells. Common antigens targeted are Clusters of Differentiation (CDs). These are specific to a certain type of cell. If you can identify which CD is presented on your cells of interest, then you can stain your sample with a fluorescent dye specific to it and using FACS, sort out only those cells. However, there are many other mechanisms by which fluorescent dyes can act.

Some dyes are able to diffuse across membranes. By taking advantage of this property of the dye, users can characterize intracellular activity as well as surface-expression of proteins. For example, in dead cells, Propidium Iodide (PI) can penetrate the nucleus where it binds to DNA. The fluorescent signal of PI can be used to quantify DNA content for cell cycle analysis or to identify dead cells in a sample.

Certain fluorescent dyes can be used to characterize kinetic intracellular activity rather than fixing cells in formaldehyde and losing viable cells. The table below outlines dyes that can be used to measure several parameters of cytotoxicity caused by oxidative stress.

Dye Parameter Mechanism of Action Excitation/ Emission
DCFH-DA Radical Oxidative Species

(ROS)

Deacetylated to

2’7’ dichlorofluorescin which reacts with ROS under radical conditions to 2’7 dichlorofluorescein (DCF)

488nm/525nm
Rh123 Mitochondria Membrane Potential

(MMP)

Sequestered by active mitochondria 488nm/525nm
Indo-1 AM Calcium Levels Emits at two different wavelengths depending on presence of calcium ions 350nm/[400nm/485nm]
PI Live/Dead Permeates dead cells only and binds to DNA 488nm/675nm

This experimental setup is just one example of the capability of flow cytometry. In FACS systems, these characterized cells can then be sorted and purified for further experiments.

Cell Sorting

  1. Fluorescence-Activated Cell Sorting Analysis of Heterotypic Cell-in-Cell Structures[1]
  2. Fluorescence-Activated Cell Sorting[2]
  3. Flow Cytometry: First Principles[3]
  4. Flow Cytometry and Cell Sorting[4]
  5. Table of Fluorescent Dyes used for Common CD markers[5]
  6. Kinetic Characterization of Intracellular Activity[6]
  1. ^ He, Meifang; Huang, Hongyan; Wang, Manna; Chen, Ang; Ning, Xiangkai; Yu, Kaitao; Li, Qihong; Li, Wen; Ma, Li (2015-04-27). "Fluorescence-Activated Cell Sorting Analysis of Heterotypic Cell-in-Cell Structures". Scientific Reports. 5 (1). doi:10.1038/srep09588. ISSN 2045-2322. PMC 5386181. PMID 25913618.{{cite journal}}: CS1 maint: PMC format (link)
  2. ^ Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A. (1972-03). "Fluorescence Activated Cell Sorting". Review of Scientific Instruments. 43 (3): 404–409. doi:10.1063/1.1685647. ISSN 0034-6748. {{cite journal}}: Check date values in: |date= (help)
  3. ^ Longobardi., Givan, Alice (2001). Flow cytometry : first principles (2nd ed ed.). New York: Wiley-Liss. ISBN 0471459119. OCLC 53228572. {{cite book}}: |edition= has extra text (help)CS1 maint: multiple names: authors list (link)
  4. ^ Ibrahim, Sherrif F.; van den Engh, Ger (2007), "Flow Cytometry and Cell Sorting", Cell Separation, Springer Berlin Heidelberg, pp. 19–39, doi:10.1007/10_2007_073, ISBN 9783540752622, retrieved 2018-10-17
  5. ^ "Human CD & Other Cellular Antigens | Thermo Fisher Scientific - US". www.thermofisher.com. Retrieved 2018-12-02.
  6. ^ Sureda, Francesc X; Gabriel, Cecı́lia; Comas, Jaume; Pallàs, Mercè; Escubedo, Elena; Camarasa, Jorge; Camins, Antonio (1999-12). "Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry". Brain Research Protocols. 4 (3): 280–287. doi:10.1016/s1385-299x(99)00030-6. ISSN 1385-299X. {{cite journal}}: Check date values in: |date= (help)
Retrieved from "https://en.wikipedia.org/w/index.php?title=User:ApexofAdharma/sandbox&oldid=874069270"