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Updated: Aug 18, 2026
A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice
Published on: April 19, 2020
Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures,
Abstract:
Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Hepatocyte nuclei show higher protein fluorescence than thymocyte nuclei. Nuclear matrices and residual structures exhibit differential fluorescence, with CPM revealing unique protein characteristics in these nuclear components.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Nuclear structures and their protein composition are crucial for cellular function.
- Microfluorometry allows for the quantitative analysis of nuclear components.
Purpose of the Study:
- To compare protein characteristics of isolated hepatocyte and thymocyte nuclei and their derivatives (residual structures, nuclear matrices).
- To evaluate the utility of different fluorochromes (BSF, AO, CPM) in differentiating nuclear proteins.
Main Methods:
- Microfluorometry analysis of isolated nuclei and their derivatives (residual structures, nuclear matrices).
- Fluorochroming with brilliant sulfaflavine (BSF) for total protein, acridine orange (AO) for acidic protein groups, and CPM for sulfhydryl/disulfide groups.
- Comparative analysis of fluorescence intensity and ratios between cell types and nuclear structures.
Main Results:
- Hepatocyte nuclei and derivatives consistently showed higher fluorescence than thymocyte counterparts across all fluorochromes.
- Nuclear matrices and residual structures displayed distinct fluorescence patterns with AO and BSF (matrices < residual < intact nuclei), but CPM showed matrices > intact nuclei > residual structures.
- BSF fluorescence ratios between hepatocyte and thymocyte nuclei changed significantly during extraction, unlike AO and CPM ratios.
Conclusions:
- Proteins detected by AO and CPM are distinct from those detected by BSF.
- CPM reveals unique protein characteristics in nuclear matrices compared to residual structures.
- Differential protein composition between hepatocyte and thymocyte nuclei is evident and is altered during nuclear structure isolation.
