The effect of calcium dyes on the observed dynamics of calcium signals

ESTEFANÍA PIEGARI, LUCÍA LOPEZ, LORENA SIGAUT, SILVINA PONCE DAWSON

Congreso; Congreso Anual de la Sociedad Argentina de Neurociencias (SAN); 2011

Calcium signaling is ubiquitous across cell types. Intracellular calcium signals are observed in intact cells with a minimum disruption using calcium dyes. The most commonly used indicators fluoresce upon calcium binding, thus providing information on the calcium-bound dye concentration. Their presence affects the calcium dynamics since dyes act as calcium buffers. Cells have buffers, usually proteins, that bind calcium altering its spatio-temporal dynamics. In this work we address the issue of to what extent the observed signals vary depending on the dye that is used. To this end we perform experiments in Xenopus Laevis oocytes using confocal microscopy and two dyes, Fluo 4 and Rhod 2, to observe signals that arise due to calcium release from the endoplasmic reticulum through IP3 receptors (IP3R’s). The dyes probed differ in their binding kinetics and dissociation constant. However, we have found that it is possible to choose their concentrations in such a way that, when using separately, the properties of the observed signals remain undistinguishable. We also observe signals in the presence of both dyes using a multispectral microscope. In this way we are able to study the competition between the two dyes probing the effect of their different kinetics on the observed signals.