Hello all, I have a flow chamber to study calcium signalling in endothelial cells under exposure to fluid flow (cell culture medium). The indicator used is Fluo-3. Dye loading of 1.5 um Fluo-3 for 20 min RT is used to give low levels of intracellular dye, in order to minimise buffering of the calcium responses. I have heard that there are potential fixation effects due to the aldehyde degradation products of fluorescein-derived probes which are produced during photobleaching, which occurs during scanning. The original papers on the development of Fluo-3 and its application in living cells (JBC 1989 (264) 8171-8 & 8179-84), don't discuss the possibility of this complication (& the illumination was with a xenon lamp rather than a laser). 1. Does anyone know whether aldehyde-related fixation is likely at the concentrations of fluo-3 used for calcium measurements? What intracellular concentrations of a fluorescein-derived compounds could yield aldehydes that would give this problem? Does anyone have references discussing this problem? 2.In addition this factor was suggested as a possible reason for loss of contractility in experiments involving scanning of smooth muscle cells. Any comments? I would very much appreciate any information on this question. Thanks in advance. Natalie James ----------------------------------------------------------------------- Natalie James, PhD CSIRO Division of Biomolecular Engineering Riverside Corporate Park PO Box 184 North Ryde NSW 2113, AUSTRALIA [log in to unmask] (Ph) 61-2-886-4885 (Fax) 61-2-886-4818