Ng C E, Koch C J, Inch W R
Cell Biophys. 1980 Jun;2(2):153-63. doi: 10.1007/BF02795841.
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.
EMT6纤维肉瘤细胞在组织培养中生长至指数期，然后在37℃的低氧条件下孵育。测定浮力密度作为低氧处理时间的函数。低氧通过两种方式产生。第一种方法是将细胞置于密封的铝制培养箱中，箱内含有95%氮气、5%二氧化碳气体以及<10 ppm的氧气，细胞会迅速暴露于低氧环境。在37℃孵育后，将它们在线性Ficoll梯度中离心至等密度点。4小时后发现密度显著降低，延长孵育至24小时未导致进一步变化。这种密度变化在转回有氧条件下是可逆的，低氧细胞在空气中再孵育约10小时后恢复到有氧密度。产生低氧的第二种方法是在充满培养基的气密容器中培养约8×10⁶个细胞。随着培养基中的氧气被细胞呼吸消耗，低氧逐渐产生。得到了相似的结果，即低氧细胞的密度显著降低。然而，当细胞耗尽所有原始氧气后，每隔2小时将低氧水平在4000和<10 ppm之间变化时，低氧细胞与有氧细胞之间的密度没有显著差异。
