The application of flow cytometry to the study of nuclear matrix. A multiparametric analysis.
The nuclear matrix, which in situ corresponds essentially to the interchromatin ribonucleoprotein particles, once isolated appears constituted by a peripheral lamina connected to the nucleolar remnant by a fibrous network. The features of the matrix components depend on the procedure employed during its purification, a multistep method commonly involving nuclease digestion, low and high salt extractions and treatment with a non ionic detergent. Here is reported the application of flow cytometry to the analysis of matrix purification. The experimental data indicate that, despite deep changes of the nuclear content due to the selective extraction of almost all nucleohistone and membrane components, the scatter pattern of the matrix, which is drastically lowered as the nucleic acid content decreases, closely resembles that of the starting nuclei. This behaviour permits one to follow the different matrix populations deriving from diploid and polyploid parenchymal liver nuclei throughout the isolation procedure and confirms the feasibility of flow cytometry for both analysis and sorting of nuclei and nuclear matrices. In addition, an ultrastructural analysis on thin sections and a morphometric study by means of semiautomated image analysis of phase contrast micrographs have been performed. The image analysis showed a decrease in particle dimension to about 50% of the nuclear value, which could partially explain the reduction of forward light scatter.