Continuous Measurement of Changes in Intracellular Calcium Concentration in Mouse Splenic T Cells Attached to a Glass...

Mitogen- and isoproterenol-induced changes of [Ca(2+)](i) in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak((R)) (3.5 &mgr;g/cm(2)). The cells were then loaded with fluorescent dye (2 &mgr;g/ml of fura2-AM or fluo3-AM) and changes in [Ca(2+)](i) in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca(2+)](i) demonstrated that concanavalin A (Con A, 2 or 5 &mgr;g/ml) caused an increase in [Ca(2+)](i) that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05-5 &mgr;g/ml) had an EC(50) of approximately 0.3 &mgr;g/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca(2+)](i). In single cell studies, the increase in [Ca(2+)](i) in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together these data demonstrate that Cell Tak((R)) can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells. Copyright 1995 S. Karger AG, Basel