title: Selective fractionation of hypoxic B16 melanoma cells by density gradient centrifugation.
abstract: Cells of the mouse B16 melanoma growing in monolayer culture and as tumors were fractionated by isopycnic density centrifugation in a linear-density (1.02-1.20 g/ml) metrizamide gradient. Cultured cells concentrated into one or two distinct bands, with densities of 1.02-1.04 g/ml and 1.06-1.10 g/ml, depending on growth conditions. Cells subjected to extreme hypoxia (less than 0.02% O2) banded predominantly at the lower density, and normally-oxygenated cells banded at the higher density. Fractionated tumor cells concentrated at both densities. Compared with cells at the higher density, lower-density cells incorporated more of the hypoxic cell radiosensitizer [14C]misonidazole and less [3H]thymidine in vivo, were less clonogenic but more resistant to X-irradiation in situ, and labeled to a lesser extent with intravenously-delivered Hoechst 33342 fluorochrome, a marker for cells proximal to tumor blood vessels. Lower-density tumor cells were, therefore, enriched in non-proliferating radioresistant hypoxic cells from tumor regions remote from blood vessels.
authors.affiliation: Department of Pathology, New York Medical College, Valhalla 10595.
authors.forename: M
authors.initials: M
authors.lastname: Fornabaio
authors.forename: Y S
authors.initials: YS
authors.lastname: Kim
authors.forename: C W
authors.initials: CW
authors.lastname: Stackpole
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D008546:Melanoma, Experimental / Q000473:pathology*; D008793:Metrizamide / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen; D014407:Tumor Cells, Cultured
chemical_list: D008793:Metrizamide; D010100:Oxygen