Astrocyte differentiation in primary culture followed by flow cytometry

Astrocyte proliferation and differentiation in primary culture were followed by flow cytometry. The time-courses of the percentages of astrocytes in the different cell-cycle phases suggest that astrocytes proliferate during the first 10 days in culture thereafter reaching confluence. Two types of astrocytes are identified immunocytochemically: one growing in the bed monolayer, identified as type-1 astrocytes, and another growing on the top of the monolayer, identified as type-2 astrocytes. In addition, three populations identified as being formed of type-1, type-2 and putative progenitor cells, respectively, were followed by flow cytometry. Progenitor cells were the major type 2 h after plating (89%) but their percentage decreases sharply (to 16%) during the first 5 days in culture, with no ensuing changes. In contrast, the percentage of type-1 cells (11%) rapidly increased after plating reaching a maximum 5 days later (73%). Later, it decreased (to 47%) and was maintained thereafter. The percentage of type-2 cells was undetectable immediately after plating but increased from the 3rd to the 10th day with no further changes. Our results suggest that progenitor cells differentiate into type-1 astrocytes triggered by the culture medium but the differentiation of progenitor cells into type-2 astrocytes is brought about by some type-1-secreted factor. In this work we report a rapid and simple method for following the growth and differentiation of rat brain astrocytes in primary culture.