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 t o follow-zip S phase, in relation to DNA content, in order to assess the se ssed 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%. CONCLUSION: Quantitative fluorescence imaging allows a sensitive determinat ion of DNA content for adherent-cell nuclei in situ. Topologic analyses of nuclear components will be possible in relation to DNA content.precision of DNA measurements. STUDY DESIGN: Double labeling experiments involved, total DNA staining with Hoechst 33342 and BrdU immunostaining (after either Bu photolysis and DNA strand break labeling by terminal transferase or acid denaturation) to dete ct replicating DNA. An epifluorescence microscope was used, images captured with a CCD camera and quantitative total DNA measurements dor re in 12 bit s with IPLab software. BrdU results were related to DNA content on an indiv idual cell basis. Cell cycle analyses mere run with Imastat software (devel oped in the laboratory) on Hoechst-stained cells and on double labeled cell s. RESULTS: In cells progressing through the cycle, as assessed by BrdU, a cor responding increase in DNA content was measured. Early S differed from G1 ( P<.05). Imastat analyses gave a CV for GI peak of 6-7%. CONCLUSION: Quantitative fluorescence imaging allows a sensitive determinat ion of DNA content for adherent-cell nuclei in situ. Topologic analyses of nuclear components will be possible in relation to DNA content.