LOCAL CHANGES OF MEDIUM IN STUDIES OF INDIVIDUAL CELLS

A procedure is described for changing the medium surrounding individua l 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 med ium. The medium to be applied was perfused through the U-tube by press ure at one end and suction at the other. To prevent premature delivery of new medium from the U-tube, suction of the outlet dominated result ing in a net withdrawal of medium from the cell chamber. The flow of m edium through the hole could be reversed rapidly by arresting the suct ion with an electromechanical valve. In this way it was possible to ob tain 95% replacement of medium within 60 ms. A pressure transient aris ing from the closure of the valve was damped by the presence of a smal l air bubble in the system. To secure a precise deposition of medium a nd minimize the risk of mechanical disturbances to the cell it was ess ential to be able to inspect the medium changes visually. For this pur pose the fluorescent indicator rhodamine B bound to dextran proved sat isfactory. Free rhodamine Il could not be used because it had biologic al effects, as was evident from studying ATP-regulated K+ channels in pancreatic beta-cells. When using a purpose-designed syringe pump for perfusing the U-tube, the technique allows well controlled exposure of individual cells to test substances added together with dextran-linke d rhodamine B.