Quantitative fluorescence imaging approach for the study of polyploidization in hepatocytes.
We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms.
Lamas
Eugenia
E
Liver Cancer and Molecular Virology, Institut National de la Santé et de la Recherche Médicale, Unité 370, Paris, France.
Chassoux
Danielle
D
Decaux
Jean-François
JF
Brechot
Christian
C
Debey
Pascale
P
D000367:Age Factors; D000818:Animals; D001562:Benzimidazoles; D001973:Bromodeoxyuridine; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000648:ultrastructure*; D008297:Male; D008856:Microscopy, Fluorescence; D011123:Polyploidy; D051381:Rats; D017208:Rats, Wistar
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D004247:DNA; D001973:Bromodeoxyuridine; C017807:bisbenzimide ethoxide trihydrochloride