The ability of neonatal astrocytes to promote neurite outgrowth in vitro and in vivo diminishes as astrocytes mature. This property correlates with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine differences between these two populations. Previous work on immortalization of bipotential neural/glial precursors and fully differentiated glial cells suggests that immortalization of astrocytes at timed intervals of culture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by retrovirus-mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact-inhibited. Three phenotypes that change as a function of astrocyte maturation were examined to determine the fidelity with which the cell lines represent immature and mature astrocytes. These were: (1) cell morphology, growth pattern and size, (2) level of glial fibrillary acidic protein (GFAP) expression, and (3) neurite outgrowth promotion. First, immature and mature lines resemble mortal type 1 astrocytes of corresponding ages with respect to morphology and growth pattern, and retain a quantitative difference in cell size (mature cells are larger). Second, the pattern of GFAP expression is preserved, with immature lines expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was examined. While all neurite outgrowth measures are significantly greater for the immortalized lines than for control 3T3 cells, they are attenuated relative to mortal astrocytes. The age-related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initiation, but not retained for mean neurite length. Thus, SV40 Tag-immortalized astrocytes have a complex phenotype characterized by retention of age-related differences in morphology, growth pattern and cell size, and by a marked attenuation of some astrocyte-specific characteristics but retention of age-related differences in the expression level of these same characteristics, and finally, loss of the ability to support neurite extension at level characteristic of immature astrocytes.

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A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 p g / d to 5 pg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed. o 1992 Wiey-Liss, Inc.Keyterms: SV40, large T antigen, indirect immunofluorescence, propidium iodide, multiparameter analysis Several reports (see references in 9, 10, and 15) have supported the view that an in-depth understanding of the mechanism of action of cell proliferation-regulating genes could be facilitated by flow cytometric measurement of their products as a function of the cell cycle. This can be achieved by dual-parametric analysis of gene expression and DNA content through simultaneous immunofluorescence and propidium staining. The fixation and staining techniques have worked well for quantification of abundant or moderately abundant intracellular antigens (9,lO). However, current techniques have limited sensitivity that makes analysis of low-abundance antigens difficult (e.g., ref. 13).Various cytometric studies have described techniques that can sensitively measure cell-surface antigens (copy number of a few hundred molecules) by indirect immunofluorescent labeling (7,27). Intracellular antigens have been less amenable to sensitive analysis (5,6,11,17). Besides background fluorescence, the limiting factors for antigen detection are incomplete permeabilization, non-specific binding of antibody, and propidium emission below 540 nm.One way to increase the sensitivity of a labeling assay is through further signal amplification by introducing additional steps of multilayered immunolabeling (19). The high affinity of biotin for streptavidin (Kd -M) has been utilized to amplify immunohistochemical staining using biotinylated secondary antibody and labeled streptavidin (16), in enzyme-linked immunosorbent assays (ELISA) (25) and for membrane receptor assays (12,22,26,27). Fluorochrome-labeled streptavidin has been used for flow cytometric detection of cell-surface antigens (2,12,26), RNA (3), and DNA polymerase ci (23).With the goal of gaining an insight into mechanisms of normal and abnormal growth regu...

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