Density Centrifugation of Murine Fibrosarcoma Cells Following In Situ Labelling With Tritiated Thymidine

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Abstract
Murine fibrosarcoma (FSa) cells form at least 5 unique subpopulations after centrifugation in linear Renografin density gradients. Each of these subpopulations was characterized with respect to selected kinetic parameters using pulse-labeling techniques and flow microfluorometry (FMF) analysis. Tumor suspensions were separated by density gradient centrifugation. Each gradient was fractionated and the density, cell number, tritium activity and labeling index (LI) per fraction were determined. There was a higher uptake of [3H]TdR in the cells recovered at the lighter (1.06-1.12 g/cm3) as compared to the heavier (> 1.12 g/cm3) densities. The FMF profiles describing the DNA contents of the cells banding in the gradient showed no difference in proportion of S-phase cells among the separated subpopulations. This lack of correlation between the FMF determination of S-phase cells and labeling index for the denser cell populations implies that DNA content alone is not an effective measurement of the functional activity of cells in solid tumors. The relatively reduced uptake of [3H]TdR by these denser cells suggests that they may have resided at relatively large distances from the functional vasculature in the tumor.