Abstract
A procedure is described for changing the medium surrounding individual cells attached to the bottom of a cell chamber. A small hole at the "apex" of a plastic U-tube allowed application and withdrawal of medium. The medium to be applied was per fused through the U-tube by pressure at one end and suction at the other. To prevent premature delivery of new medium from the U-tube, suction of the outlet dominated resulting in a net withdrawal of medium from the cell chamber. The flow of medium through the hole could be reversed rapidly by arresting the suction with an electromechanical valve. In this way it was possible to obtain 95% replacement of medium with in 60 ms. A pressure transient arising from the closure of the valve was damped by the presence of a small air bubble in the system. To secure a precise deposition of medium and minimize the risk of mechanical disturbances to the cell it was essential to be able to inspect the medium changes visually. For this purpose the fluorescent indicator rhodamine B bound to dextran proved satisfactory. Free rhodamine B could not be used because it had biological effects, as was evident from studying ATP-regulated K+ channels in pancreatic β-cells. When using a purpose-designed syringe pump for per fusing the U-tube, the technique allows well controlled exposure of individual cells to test substances added together with dextran-linked rhodamine B.
