Scanning microfluorometric measurements of acriflavine-Feulgen-SITS-stained fibroblasts during G0-G1 transition.

For the characterization of nonproliferating cells, scanning microfluorometric measurements of mouse fibroblasts (L-929) during G0-G1 transition were carried out. Samples were taken at different time intervals after serum stimulation. Cells were stained for DNA using the acriflavine-Feulgen method and for protein with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS). Considering that acid hydrolysis removes basic proteins, SITS fluorescence represents acidic proteins, which within the nucleus are to a large degree located in the nucleoli. From each preparation, nuclei were scanned at a 0.5 micrometer step size, measuring DNA and protein fluorescence successively. Fluorescence data of nucleoli were evaluated. The number of nucleoli reached a maximum two hours after stimulation. Both the total nucleolar area and fluorescence were found to increase, up to 8 and 11 hours, respectively, by a factor of four to five. This indicated that these fluorescence parameters can be used to distinguish between resting and cycling cells.