A reaction vessel for the measurement of oxygen consumption by small numbers of cells in suspension

Abstract

A reaction vessel is described that permits measurement of oxygen consumption in small numbers of cells in suspension. The assay permits accurate detection of changes in oxygen tension amounting to less than 0.016 nmol·min −1, and allows the continuous monitoring and calculation of oxygen consumption by as few as 3−6×10 5 blood granulocytes or mouse peritoneal macrophages. The results obtained with this assay are highly reproducible, and the values agree closely with those obtained in 10–15-fold larger samples with commercially available oxygraphs. The reaction vessel offers three basic advantages: (1) fewer cells are required per sample (at most 1 10 the number needed for conventional oxygraphs); (2) smaller volumes of test media are needed (i.e., cytokines, opsonins); and (3) the oxygen tension of the medium can be rapidly changed to any desired level at the start of or during experiments. The applications described include the oxygen consumption by human granulocytes and murine peritoneal macrophages before and after addition of both soluble and particulate stimuli. With the use of this technique it was found that the maximum oxygen consumption by human granulocytes after stimulation with phorbol myristate acetate is proportional to the oxygen tension of the medium during the addition of the triggering agent.