Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures

Growth control is investigated in detail in fed and unfed HeLa‐S3 suspension cultures. Two‐step acridine orange staining and flow cytometric analysis indicate declines in cellular red fluorescence (proportional to RNA content) of 40–50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with G1‐DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1‐phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H‐thymidine pulse‐labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1‐arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 34% of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell‐cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.