DNA content by in situ fluorescence imaging and S-phase detection, with chromatin structure preserved.
OBJECTIVE
To test the feasibility of in situ DNA quantitation of adherent cells' nuclei by fluorescence imaging, preserving chromatin structure and to follow-up S phase, in relation to DNA content, in order to assess the precision of DNA measurements.


METHODS
Double labeling experiments involved total DNA staining with Hoechst 33342 and BrdU immunostaining (after either Br photolysis and DNA strand break labeling by terminal transferase or acid denaturation) to detect replicating DNA. An epifluorescence microscope was used, images captured with a CCD camera and quantitative total DNA measurements done in 12 bits with IPLab software. BrdU results were related to DNA content on an individual cell basis. Cell cycle analyses were run with Imastat software (developed in the laboratory) on Hoechst-stained cells and on double labeled cells.


RESULTS
In cells progressing through the cycle, as assessed by BrdU, a corresponding increase in DNA content was measured. Early S differed from G1 (P < .05). Imastat analyses gave a CV for GI peak of 6-7%.


CONCLUSIONS
Quantitative fluorescence imaging allows a sensitive determination of DNA content for adherent-cell nuclei in situ. Topologic analyses of nuclear components will be possible in relation to DNA content.
Chassoux
D
D
Institut National de la Recherche Agronomique Unité 806/Equipe Associée 2703 Muséum National d'Histoire Naturelle, Paris, France. chassoux@ibpc.fr
Franchi
J
J
Cao
T T
TT
Debey
P
P
D016475:3T3 Cells; D000818:Animals; D001562:Benzimidazoles; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D004261:DNA Replication; D005456:Fluorescent Dyes; D019044:Image Cytometry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D016196:S Phase / Q000235:genetics*
D001562:Benzimidazoles; D002843:Chromatin; D005456:Fluorescent Dyes; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride