We have used mixtures of Fluo-3 and Fura-Red in isolated cardiac myocytes as well as in others cells. While loading is quite variable with the AM-ester technique, dialysis via a patch-clamp electrode produces very consistent and reproducible results. As pointed out by Stepen Cody, signals from AM-loaded cells cannot be calibrated quantitatively because of these problems. If you are interested in calibrated Ca signals you need to patch the cells for loading with the salt form of the indicators. We also found significant binding and changes of the dye properties in-vivo, suggesting that a in-vivo calibration for the mixture has to be performed (Lipp, Luescher, Niggli, Cell Calcium, in press). I hope this helps Ernst Niggli Department of Physiology University of Bern Buehlplatz 5 3012 Bern Switzerland [log in to unmask]