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FLUORESCENT-PROTEIN STAINING OF NUCLEOLI IN MONOLAYER CELL CULTURES: DETERMINANTS AND SIGNIFICANCE
1 Department of Pathology, University of Colorado Medical Center, Denver, Colorado 80220
In order to distinguish whether fluorescent-protein staining of nucleoli in acetone-fixed cell culture monolayers is a useless artifact or whether nucleolus-globulin affinity reflects a physiologic interaction between cells and serum, the phenomenon was investigated by further characterizing the interacting components and the conditions required. Staining proved to be a time-dependent and temperature-dependent, two-step procedure: initial modification of nucleoli by smaller serum 
-globulins is required for later ionic binding to nucleoli of fluorescein-labeled, nonimmune 
-, 
- or larger -globulins. The initial modification is essential only in cells grown in minimal serum and harvested toward the end of the logarithmic growth phase of the culture. The nucleolar component binding the conjugate is both heat- and pH-stable, is maintained in situ apparently by ionic bonds and most likely includes a saline-insoluble, nonhistone basic protein. Previous serum modification of nucleoli in living primarily explanted monkey kidney cells, detected with this technique, could not be demonstrated with established H-Ep 2 or HeLa cell lines of human tumor origin. These data suggest that serum-dependent nucleolar fluorescent protein staining might be a useful artifact, signifying something about nucleolar organization or content of unexplained relation to cell growth.
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