Document Detail


Analysis of apoptosis by laser scanning cytometry.
MedLine Citation:
PMID:  10082299     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.
Authors:
E Bedner; X Li; W Gorczyca; M R Melamed; Z Darzynkiewicz
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.; Review    
Journal Detail:
Title:  Cytometry     Volume:  35     ISSN:  0196-4763     ISO Abbreviation:  Cytometry     Publication Date:  1999 Mar 
Date Detail:
Created Date:  1999-05-25     Completed Date:  1999-05-25     Revised Date:  2013-01-16    
Medline Journal Info:
Nlm Unique ID:  8102328     Medline TA:  Cytometry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  181-95     Citation Subset:  IM    
Affiliation:
Brander Cancer Research Institute, New York Medical College, Valhalla, USA.
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MeSH Terms
Descriptor/Qualifier:
Annexin A5 / metabolism
Apoptosis*
Cell Membrane / metabolism
Cells, Cultured
DNA / analysis
DNA Fragmentation
Flow Cytometry
Humans
Image Cytometry / methods*
Membrane Potentials
Mitochondria / metabolism
Grant Support
ID/Acronym/Agency:
CA 28704/CA/NCI NIH HHS; R01 CA028704/CA/NCI NIH HHS; R01 CA028704-20/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Annexin A5; 9007-49-2/DNA

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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