The DNA content of mouse two-cell embryos can be measured by microfluorimetric image analysis under conditions of cell viability.
Video-enhanced fluorescence imaging was used to quantify the DNA content in live two-cell mouse embryos. DNA was stained with the vital fluorophore Hoechst 33342. Conditions of dye concentration and irradiation were such that two-cell embryos could be kept in the constant presence of the dye for about 24 h without a major effect on their furtherin vitro viability. Total nuclear fluorescence intensity of stained two-cell embryos was measured twice under these conditions, i.e., in G1 (1 h after cleavage) and in G2 (15-18 h after cleavage), by image analysis. After correcting for the fluctuations in excitation intensity and for the spatial nonhomogeneities of the optical system (lenses and sensor), the mean total nuclear fluorescence intensity was about twofold higher in G2 than in G1 ([Symbol: see text]R[Symbol: see text]=1.99 to 2.25), and this increase was abolished by the addition of aphidicolin, an inhibitor of replication. The fluorescence increase did not depend on the Hoechst concentration in the range of 10-40 ng/ml, i.e., in the range of embryo viability. The coefficient of variation of the total nuclear fluorescence intensity measured under these conditions was rather large (10 to 20%). Nevertheless, the mean value of fluorescence intensity in G1 of nuclei of a given pool represents an appropriate reference to measure the increase in fluorescence intensity between G1 and G2.
Unité de Biologie de Développement, Département de Physiologie Animale, INRA, 78352, Jouy en Josas Cedex, France.
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Adenot
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Corteggiani
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Debey