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User:Logan Elkins/Viability assay

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(exact text current portion) - 3/2/2020

A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover viability. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quantifiable index between 0 and 1 (or 0% and 100%). Viability can assay mechanical activity, motility (spermatozoa or granulocytes), contraction (muscle tissue or cells), mitotic activity, etc.

For example, examining the ratio of potassium to sodium in cells can serve as an index of viability. If the cells do not have high intracellular potassium and low intracellular sodium, then (1) the cell membrane may not be intact, and/or (2) the sodium-potassium pump may not be operating well.  Unlike the potassium-sodium ratio assay, some assays do not kill cells, such as neutral red uptake.

As with many kinds of viability assays, quantitative measures of physiological function do not indicate whether damage repair and recovery is possible. An assay of the ability of a cell line to adhere and divide may be more indicative of incipient damage than membrane integrity. Fluorescent-based assays do not require large sample sizes. Viability assays are used to assess the success of cell culture techniques, cryopreservation techniques, the toxicity of substances, or the effectiveness of substances in mitigating effects of toxic substances.

Classification[edit]

[edit]
  1. Cytolysis or membrane leakage assays: This category includes the lactate dehydrogenase assay, a stable enzyme common in all cells which can be readily detected when cell membranes are no longer intact. Examples:
    1. Propidium iodide
    2. Trypan blue
    3. 7-Aminoactinomycin D
  2. Mitochondrial activity or caspase assays: Resazurin and Formazan (MTT/XTT) can assay for various stages in the apoptosis process that foreshadow cell death.
  3. Functional assays: Assays of cell function will be highly specific to the types of cells being assayed. For example, motility is a widely used assay of sperm cell function. Gamete survival can be used to assay fertility, in general. Red blood cells have been assayed in terms of deformability, osmotic fragility, hemolysis, ATP level, and hemoglobin content. For transplantable whole organs, the ultimate assay is the ability to sustain life after transplantation, an assay which is not helpful in preventing transplantation of non-functional organs.
  4. Genomic and proteomic assays: Cells can be assayed for activation of stress pathways using DNA microarrays and protein chips.
  5. Flow cytometry assays: Automation allows for analysis of thousands of cells per second.

List of common viability assays[edit]

[edit]



(draft portion) - 3/2/2020

A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover a state of survival. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quantifiable index that ranges between the integers of 0 and 1 or, if more easily understood, the range of 0% and 100%.[1] Viability can be observed through the physical properties of cells, tissues, and organs. Some of these include mechanical activity, motility, such as with spermatozoa and granulocytes, the contraction of muscle tissue or cells, mitotic activity in cellular functions, and more.[1] Viability assays provide a more precise basis for measurement of an organism's level of vitality.


Viability assays can lead to more findings than the difference of living versus nonliving. These techniques can be used to assess the success of cell culture techniques, cryopreservation techniques, the toxicity of substances, or the effectiveness of substances in mitigating effects of toxic substances.[2]


Common Viability Assay Methods

This plated viability assay measures various yeast viability though a method called "frogging". The research is completed through drop-inoculation techniques. Research has since been conducted on "tadpoling", which is a variation of "frogging" that is completed by keeping the test cells diluted in liquid throughout their examination.[3]

Though simple visual techniques of observing viability can be useful, it can be difficult to really measure an organism's/part of an organism's make-up's viability merely using the observation of physical properties. However, there are a variety of common protocols utilized for further observation of viability using assays.

Tetrazolium Reduction Assay

One useful way to locate and measure viability is to complete a Tetrazolium Reduction Assay. The tetrazolium aspect of this assay, which utilizes both positive and negative charges in its formula, promotes the distinction of cell viability in a specimen.[4]

Resazurin Reduction Assays

Resazurin Reduction Assays perform very closely to that of a tetrazolium assay, except they use the power of redox to fuel their ability to represent cell viability.[4]

Protease Viability Marker Assay

One can look at protease fuction in specimens if they wish to target viability in cells; this practice in research is known as "Protease Viability Marker Assay Concept". The actions of protease cease once a cell dies, so a clear-cut line is drawn in determining cell viability when using this technique.[4]

ATP Assay

ATP is a common energy molecule that many researchers carry extensive knowledge about, thus carrying over to how one understands viability assays. The ATP Assay Concept is a well-known technique for determining the viability of cells using the assessment of ATP and a method known as "firefly luciferase".[4]

Sodium-Potassium Ratio Assay

Another kind of assay practices the examination of the ratio of potassium to sodium in cells to serve as an index of viability. If the cells do not have high intracellular potassium and low intracellular sodium, then (1) the cell membrane may not be intact, and/or (2) the sodium-potassium pump may not be operating well.[5][6]  

Cytolysis or Membrane Leakage Assays

This category includes the lactate dehydrogenase assay. Assays such as these contain a stable enzyme common in all cells that can be readily detected when cell membranes are no longer intact. Examples of this type of assay include propidium iodide, trypan blue, and 7-Aminoactinomycin D.

Mitochondrial Activity or Caspase Assays

Resazurin and Formazan (MTT/XTT) can assay for various stages in the apoptosis process that foreshadows cell death.

Functional Assays

Assays of cell function will be highly specific to the types of cells being assayed. For example, motility is a widely used assay of sperm cell function. Gamete survival can generally be used to assay fertility. Red blood cells have been assayed in terms of deformability, osmotic fragility, hemolysis, ATP level, and hemoglobin content.[7] For transplantable whole organs, the ultimate assay is the ability to sustain life after transplantation, an assay which is not helpful in preventing transplantation of non-functional organs.[8]

Genomic and Proteomic Assays

Cells can be assayed for activation of stress pathways using DNA microarrays and protein chips.

Flow Cytometry Assays

Automation allows for analysis of thousands of cells per second.[9]

Frogging and Tadpoling

"Frogging" is a type of viability assay method utilizes an agar plate for its environment and consists of plating serial dilutions by pinning them after they have been diluted in liquid. Some of its limitations include that it does not account for total viability and it is not particularly sensitive to low-viability assays; however, it is known for its quick pace.[10] "Tadpoling", which is a method practiced after the development of "frogging", is similar to the "frogging" method, but its test cells are diluted in liquid and then kept in liquid through the examination process. The "tadpoling" method can be used to measure culture viability accurately, which is what depicts its main separation from "frogging".[10]


As with many kinds of viability assays, quantitative measures of physiological function do not indicate whether damage repair and recovery is possible.[11] An assay of the ability of a cell line to adhere and divide may be more indicative of incipient damage than membrane integrity.[12]


Extended List of Viability Assay Methods

  1. ^ a b Pegg, D. E. (1989-06-01). "Viability assays for preserved cells, tissues, and organs". Cryobiology. 26 (3): 212–231. doi:10.1016/0011-2240(89)90016-3. ISSN 0011-2240.
  2. ^ "Overview of Probes for Cell Viability, Cell Proliferation and Live-Cell Function—Section 15.1 - US". www.thermofisher.com. Retrieved 2020-03-04.
  3. ^ Welch, Aaron Z.; Koshland, Douglas E. (2013). "A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability". Yeast. 30 (12): 501–509. doi:10.1002/yea.2989. ISSN 1097-0061.
  4. ^ a b c d Niles, Andrew L.; Moravec, Richard A.; Worzella, Tracy J.; Evans, Nathan J.; Riss, Terry L. (2013-03-04), "High-Throughput Screening Assays for the Assessment of Cytotoxicity", High-Throughput Screening Methods in Toxicity Testing, John Wiley & Sons, Inc., pp. 107–127, ISBN 978-1-118-53820-3, retrieved 2020-03-03
  5. ^ Lindner, Buko; Seydel, Ulrich (1983). "Mass Spectrometric Analysis of Drug-induced Changes in Na+ and K+ Contents of Single Bacterial Cells". Microbiology,. 129 (1): 51–55. doi:10.1099/00221287-129-1-51. ISSN 1350-0872.{{cite journal}}: CS1 maint: extra punctuation (link) CS1 maint: unflagged free DOI (link)
  6. ^ Pichugin, Yuri; Fahy, Gregory M.; Morin, Robert (2006-04-01). "Cryopreservation of rat hippocampal slices by vitrification". Cryobiology. 52 (2): 228–240. doi:10.1016/j.cryobiol.2005.11.006. ISSN 0011-2240.
  7. ^ Henkelman, Sandra; Lagerberg, Johan W. M.; Graaff, Reindert; Rakhorst, Gerhard; Oeveren, Willem Van (2010). "The effects of cryopreservation on red blood cell rheologic properties". Transfusion. 50 (11): 2393–2401. doi:10.1111/j.1537-2995.2010.02730.x. ISSN 1537-2995.
  8. ^ Southard, James H. (1989-06-01). "Viability assays in organ preservation". Cryobiology. 26 (3): 232–238. doi:10.1016/0011-2240(89)90017-5. ISSN 0011-2240.
  9. ^ Davey, Hazel M. (2011-08-15). "Life, Death, and In-Between: Meanings and Methods in Microbiology". Applied and Environmental Microbiology. 77 (16): 5571–5576. doi:10.1128/AEM.00744-11. ISSN 0099-2240. PMC 3165249. PMID 21705550.{{cite journal}}: CS1 maint: PMC format (link)
  10. ^ a b Welch, Aaron Z.; Koshland, Douglas E. (2013). "A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability". Yeast. 30 (12): 501–509. doi:10.1002/yea.2989. ISSN 1097-0061.
  11. ^ Crutchfield, Alexandra; Diller, Kenneth; Brand, Jerry (1999-02-01). "Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta)". European Journal of Phycology. 34 (1): 43–52. doi:10.1080/09670269910001736072. ISSN 0967-0262.
  12. ^ Wusteman, Monica C; Pegg, David E; Robinson, Martin P; Wang, Li-Hong; Fitch, Paul (2002-02-01). "Vitrification media: toxicity, permeability, and dielectric properties". Cryobiology. 44 (1): 24–37. doi:10.1016/S0011-2240(02)00002-0. ISSN 0011-2240.
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