Photodegradation of indo-1 and its effect on apparent Ca2+ concentrations
Abstract
Fluorescent indicators that show alterations in excitation and/or emission spectra in response to changes in [Ca2+] are widely used for quantitative cytosolic [Ca2+] measurements. There are several reports of changes in apparent [Ca2+] due only to illumination, however. These results have been attributed either to photodamage to the cells or to photodegradation of the indicator. Light-induced alteration in the behavior of the dye or cells would severely hamper the interpretation of experimental data. We examined this phenomenon in indo-1 loaded cells using confocal laser scanning microscopy. Illumination of indo-1 loaded GH3 cells leads to a decrease in apparent basal [Ca2+] and decreased peaks after depolarization with KCl. When cells were double loaded with indo-1 and fluo-3, the effect of UV illumination was noticed only with the former dye. UV irradiation of indo-1 in simple buffers caused overall photobleaching and conversion to a fluorescent but Ca2+-insensitive species. The latter effect cannot be canceled by ratiometric calibration and is due to loss of carboxymethyl groups from the anilino nitrogens. This photodegradation was inhibited by extracellular administration of 10 to 100 microM Trolox, a water-soluble vitamin ...Continue Reading
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