Document Detail


A simple and rapid slide blot method to quantify cytokine-mRNA in small numbers of lymphocytes.
MedLine Citation:
PMID:  1765664     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
In this study we describe an in situ hybridization protocol suitable for the measurement of interleukin mRNAs in small numbers of cells in suspension. In this procedure defined numbers of such cells are coated onto defined areas of microscopic slides using LAB-TEK tissue culture chamber slides. 32P-labelled oligonucleotides are then hybridized to them in situ. Binding of the probes is measured by autoradiography using X ray film followed by densitometry (slide blot) or, as in standard in situ protocols, by exposure to photoemulsion. In experiments designed to demonstrate the specificity and feasability of this protocol, peripheral blood T cells were activated with mitogenic stimuli and oligonucleotide probes specific for IL-2, IFN-gamma, or a control probe (IL-2 random) were hybridized to them. Both lymphokine genes were found to be transcribed in two discrete phases with the first peak at 12 h and another peak 48 h after stimulation, as measured semi-quantitatively on a per cell basis using X ray film exposure. The proposed experimental protocol supports the advantages of X ray film autoradiography such as rapidity, simplicity and the objectivity of densitometric evaluation, but also permits microscopic evaluation of individual cells as performed in classical in situ hybridization protocols using photoemulsion.
Authors:
M Schwarz; S Weber; J M Seifert; W Holter
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of immunological methods     Volume:  145     ISSN:  0022-1759     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  1991 Dec 
Date Detail:
Created Date:  1992-02-14     Completed Date:  1992-02-14     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  NETHERLANDS    
Other Details:
Languages:  eng     Pagination:  27-32     Citation Subset:  IM    
Affiliation:
Vienna Int. Research Coop. Center (VIRCC), University of Vienna, Austria.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Cytokines / genetics*
Gene Expression
Humans
Interferon-gamma / genetics
Interleukin-2 / genetics
Lymphocytes / chemistry*
Molecular Sequence Data
Nucleic Acid Hybridization
Oligonucleotide Probes
RNA, Messenger / analysis*
Tumor Cells, Cultured
Chemical
Reg. No./Substance:
0/Cytokines; 0/Interleukin-2; 0/Oligonucleotide Probes; 0/RNA, Messenger; 82115-62-6/Interferon-gamma

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


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