Density centrifugation of murine fibrosarcoma cells following in situ labelling with tritiated thymidine.

Abstract

Murine fibrosarcoma (FSa) cells form at least five unique subpopulations after centrifugation in linear Renografin density gradients. Each of these subpopulations has been characterized with respect to selected kinetic parameters using pulse-labelling techniques and flow microfluorometry (FMF) analysis. Tumour-bearing mice were first injected intraperitoneally with a pulse label of tritiated thymidine ([3H]TdR, 50 microCi). Following 15, 30, 60 min or 24 hr these animals were injected with cold thymidine. Animals were killed, their tumours removed and made into suspension, and separated by density gradient centrifugation. Each gradient was fractionated and the density, cell number, tritium activity, and labelling index (LI) per fraction were determined. These data were then compared to FMF data for selected cell density, cell number, tritium activity, and labelling index (LI) per fraction were determined. These data were then compared to FMF data for selected cell density bands. The results indicated a relatively higher uptake of [3H]TdR in the cells recovered at the lighter (1.06-1.12 g/cm3) as compared to the heavier (greater than 1.12 g/cm3) densities. Following a 30-min pulse, the LI's of light cells (less than 1.12 g/cm3) ranged from 25 to 30%, while the heavier cells (greater than 1.12 g/cm3) had LI's between 10 and 15%. The unseparated control cells had an LI of 19%. comparable results were found at the other times tested. In contrast, 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 labelling index for the denser cell populations implies that DNA content alone is not an effective measurement of the functional activity of cells in solid tumours. Finally, 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 tumour.