A spectrophotometric method for measuring oxygen consumption in monolayers of cultured endothelial cells

A new method for measuring oxygen consumption in monolayer cell cultures is described. Cells are cultured in shallow microchambers etched in glass chips. A haemoglobin solution is added and the microchambers are sealed airtight, the oxygenated haemoglobin solution serving as an indicator of oxygen tension within the microchamber as well as a source of oxygen. With cellular extraction of oxygen, an increasing amount of reduced haemoglobin is formed within the microchamber resulting in an increased absorbance of the haemoglobin solution at 435 nm. With a specially prepared device, it is possible to record this absorbance change with an ordinary laboratory spectrophotometer and, from this recording, it is possible to calculate the respiratory rate, provided the number of cells (or any other quantity of biological material), microchamber volume and haemoglobin concentration are known. Repeated measurements on the same undisturbed culture is possible. The system was tested on monolayer cultures of endothelial cells from the rat pulmonary artery.