Linked Life Data

16081806

Source:http://linkedlifedata.com/resource/pubmed/id/16081806

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2005-8-5
pubmed:abstractText
Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
AIM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0022-1767
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
175
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2357-65
pubmed:dateRevised
2011-11-17
pubmed:meshHeading
pubmed-meshheading:16081806-Animals, pubmed-meshheading:16081806-Antigens, Surface, pubmed-meshheading:16081806-Binding Sites, Antibody, pubmed-meshheading:16081806-Biological Markers, pubmed-meshheading:16081806-Cell Membrane Permeability, pubmed-meshheading:16081806-Cells, Cultured, pubmed-meshheading:16081806-Fixatives, pubmed-meshheading:16081806-Flow Cytometry, pubmed-meshheading:16081806-Fluorescent Dyes, pubmed-meshheading:16081806-Formaldehyde, pubmed-meshheading:16081806-Intracellular Fluid, pubmed-meshheading:16081806-Male, pubmed-meshheading:16081806-Methanol, pubmed-meshheading:16081806-Mice, pubmed-meshheading:16081806-Mice, Inbred BALB C, pubmed-meshheading:16081806-Mice, Inbred C57BL, pubmed-meshheading:16081806-Phosphoproteins, pubmed-meshheading:16081806-Phosphorylation, pubmed-meshheading:16081806-Staining and Labeling
pubmed:year
2005
pubmed:articleTitle
Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.
pubmed:affiliation
Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural, Validation Studies