Document Detail


The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry.
MedLine Citation:
PMID:  2424868     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Authors:
G J van den Engh; B J Trask; J W Gray
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Histochemistry     Volume:  84     ISSN:  0301-5564     ISO Abbreviation:  Histochemistry     Publication Date:  1986  
Date Detail:
Created Date:  1986-07-25     Completed Date:  1986-07-25     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0411300     Medline TA:  Histochemistry     Country:  GERMANY, WEST    
Other Details:
Languages:  eng     Pagination:  501-8     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Bisbenzimidazole / diagnostic use
Cell Line
Cell Nucleus / analysis*
Chromatin / analysis
Chromomycin A3 / diagnostic use
Chromosomes / analysis*
Citrates / diagnostic use
Citric Acid
DNA / analysis*
Energy Transfer
Flow Cytometry / methods
Fluorescent Dyes / diagnostic use*
Humans
Karyotyping / methods*
Male
Mice
Propidium / diagnostic use
Chemical
Reg. No./Substance:
0/Chromatin; 0/Citrates; 0/Fluorescent Dyes; 23491-44-3/Bisbenzimidazole; 36015-30-2/Propidium; 7059-24-7/Chromomycin A3; 77-92-9/Citric Acid; 9007-49-2/DNA

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


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