Click-iT assay with improved DNA distribution histograms.
The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.
Hamelik
Ronald M
RM
Department of Pathology, University of Miami Miller School of Medicine, Miami 33101, Florida, USA.
Krishan
Awtar
A
D001681:Biological Assay / Q000379:methods*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D013194:Staining and Labeling; D013997:Time Factors
D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine