Buoyant density of EMT6 fibrosarcoma cells: time course of the density changes after growth in hypoxia.

EMT6 fibrosarcoma cells were grown to the exponential phase in tissue culture and incubated at 37 degrees C under hypoxic conditions. Buoyant density was determined as a function of the time in hypoxia. Hypoxia was produced in two ways. The first involved incubation of the cells in sealed aluminum chambers containing 95% N2, 5% CO2 gas, and < 10 ppm oxygen, resulting in the cells rapidly becoming exposed to the hypoxic environment. After incubation at 37 degrees C, they were centrifuged in linear Ficoll gradients to their isopycnic density. A significant decrease in density was found after 4 h, and prolonged incubation up to 24 h did not result in further change. This density change was reversible on transfer back to aerobic conditions, with the hypoxic cells reverting to their aerobic density after about 10 h reincubation in air. The second method of producing hypoxia involved growing about 8 X 10(6) cells in a medium-filled air-tight container. Hypoxia was produced gradually as the oxygen in the medium was consumed by cellular respiration. Similar results were obtained; that is, hypoxic cells became significantly less dense. However, when the level of hypoxia was varied between 4000 and < 10 ppm at 2-h intervals after the cells had depleted all of the original oxygen, no significant difference in density was found between hypoxic and aerobic cells.