Linked Life Data

3965482

Source:http://linkedlifedata.com/resource/pubmed/id/3965482

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1985-2-14
pubmed:abstractText
A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0021-9541
pubmed:author
pubmed:issnType
Print
pubmed:volume
122
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
159-65
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1985
pubmed:articleTitle
Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't