Selective fractionation of hypoxic B16 melanoma cells by density gradient centrifugation

Cancer Lett. 1989 Mar;44(3):185-90. doi: 10.1016/0304-3835(89)90059-1.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Fractionation
  • Centrifugation, Density Gradient
  • Melanoma, Experimental / pathology*
  • Metrizamide / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Oxygen*
  • Tumor Cells, Cultured


  • Metrizamide
  • Oxygen