R E Rumbaut1, A J Sial. 1. Department of Internal Medicine, University of Missouri-Columbia, 65212, USA.
Abstract
OBJECTIVE: Fluorescent dyes are commonly used as probes for assessment of macromolecular permeability. Despite numerous examples of light-dye induced toxicity in the microvasculature, little is known regarding the relative phototoxicity of commonly used fluorescent conjugates. We, therefore, compared the phototoxicity of four fluorescent conjugates of bovine serum albumin (BSA) available commercially. METHODS: An in vitro photohemolysis assay was used, in which rat erythrocytes were incubated with fluorescently labeled BSA and exposed to epi-illumination, using an inverted microscope designed for microvascular permeability experiments. Photohemolysis was quantified by monitoring light transmission across the cells. RESULTS: Photohemolysis was dependent on excitation light intensity and fluorochrome concentration. Fluorescein isothiocyanate (FITC)-labeled BSA was the most phototoxic compound tested, inducing 50% of maximum response in 14 min. The relative phototoxicity of the BSA conjugates was: FITC > BODIPY-FL > Texas Red > tetramethylrhodamine isothiocyanate. The phototoxicity of FITC-BSA was related to a high molar dye content. Photohemolysis with each of the conjugates was inhibited by histidine, a singlet oxygen quencher. CONCLUSIONS: Relative phototoxicity of fluorescent albumin conjugates differs considerably. Selection of fluorescent conjugates for use in microvascular experiments based on phototoxicity should consider both the type and molar content of the fluorochrome.
OBJECTIVE: Fluorescent dyes are commonly used as probes for assessment of macromolecular permeability. Despite numerous examples of light-dye induced toxicity in the microvasculature, little is known regarding the relative phototoxicity of commonly used fluorescent conjugates. We, therefore, compared the phototoxicity of four fluorescent conjugates of bovine serum albumin (BSA) available commercially. METHODS: An in vitro photohemolysis assay was used, in which rat erythrocytes were incubated with fluorescently labeled BSA and exposed to epi-illumination, using an inverted microscope designed for microvascular permeability experiments. Photohemolysis was quantified by monitoring light transmission across the cells. RESULTS: Photohemolysis was dependent on excitation light intensity and fluorochrome concentration. Fluorescein isothiocyanate (FITC)-labeled BSA was the most phototoxic compound tested, inducing 50% of maximum response in 14 min. The relative phototoxicity of the BSA conjugates was: FITC > BODIPY-FL > Texas Red > tetramethylrhodamine isothiocyanate. The phototoxicity of FITC-BSA was related to a high molar dye content. Photohemolysis with each of the conjugates was inhibited by histidine, a singlet oxygen quencher. CONCLUSIONS: Relative phototoxicity of fluorescent albumin conjugates differs considerably. Selection of fluorescent conjugates for use in microvascular experiments based on phototoxicity should consider both the type and molar content of the fluorochrome.