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Using a procedure to stain cells simultaneously for cyclin B1 protein and DNA, we have examined cyclin B1 expression by flow cytometry in human cells under a variety of perturbing and nonperturbing conditions. The method described is useful for measuring relative differences in cyclin B level (immunochemically detectable epitope) as a function of cell cycle position on an individual cell basis and thus to examine cell cycle-related changes in cyclin B expression without prior cell synchronization. We show that in HeLaS3 cells, cyclin B1 accumulates in cells only after they become 4C and have resided in G2 for a short period of time. During colcemid-induced mitotic arrest cyclin B1 continues to accumulate in HeLa S3 cells, and under specific conditions of aphidicolin-induced unbalanced cell growth induced, cyclin B accumulates to supranormal levels prior to mitosis. Flow cytometric analysis of cyclin B expression and DNA content permits detailed examination of the effects of cell cycle perturbations on cyclin B expression under a variety of conditions.

Dissociated hippocampal cultures from fetal mice (13--18 days gestational age) can be maintained for up to two months in culture. Cells grow as either isolated neurons or in small neuronal aggregates. Neurons remain small with a soma diameter of 15--20 micrometer even in mature cultures and develop extensively branched processes during the first two weeks in culture. After this time, processes become more difficult to visualize with phase-contrast optics because of a tendency to grow within the underlying non-neuronal cells. However, the presence of processes has been proved by silver-staining which demonstrates an organizational complexity ranging from a loosely reticulated neuropil to fascicles containing many fibers. More detailed study of individual neuronal morphology was carried out in cells filled with the fluorescent dye, Lucifer Yellow CH, in conjunction with the intracellular recording of synaptic and action potentials from dye-containing micropipettes. Dye-filled cells show a well-developed branching morphology. Process specializations include spines, beading, and basket-like endings. Processes tend to emanate from one side of the soma, either originating at the cell body or from a single trunk. Commonly there are 2--4 orders of branching, but up to 6 orders can occur (counted centrifugally from the soma). Electron microscopy revealed synapses distributed predominantly on dendrites with a smaller number on somata. Dendritic spines are present and are contacted principally by asymmetric synaptic junctions. Symmetric synapses are relatively more common on somata and proximal dendrites.