Linked Life Data

9202713

Source:http://linkedlifedata.com/resource/pubmed/id/9202713

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1997-7-22
pubmed:abstractText
The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0022-1759
pubmed:author
pubmed:issnType
Print
pubmed:day
12
pubmed:volume
204
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
89-98
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:9202713-Animals, pubmed-meshheading:9202713-Antibodies, pubmed-meshheading:9202713-Chromium, pubmed-meshheading:9202713-Cytotoxicity Tests, Immunologic, pubmed-meshheading:9202713-Genes, Reporter, pubmed-meshheading:9202713-Humans, pubmed-meshheading:9202713-Hydrogen-Ion Concentration, pubmed-meshheading:9202713-Luciferases, pubmed-meshheading:9202713-Lymphocytes, pubmed-meshheading:9202713-Mice, pubmed-meshheading:9202713-Mice, Inbred C57BL, pubmed-meshheading:9202713-Nitrophenylgalactosides, pubmed-meshheading:9202713-Phenolsulfonphthalein, pubmed-meshheading:9202713-Sensitivity and Specificity, pubmed-meshheading:9202713-Spleen, pubmed-meshheading:9202713-Substrate Specificity, pubmed-meshheading:9202713-Transfection, pubmed-meshheading:9202713-Tumor Cells, Cultured, pubmed-meshheading:9202713-beta-Galactosidase
pubmed:year
1997
pubmed:articleTitle
A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines.
pubmed:affiliation
Department of Immunology, Robert Koch-Institute, Berlin, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't