Background Quantifying the amount of albumin conjugated to Evans Blue dye (Alb-EB) fluxing across organ barriers is a popular technique to measure intactness of the physical barrier in rodent models of a variety of diseases. We have reevaluated this technique in terms of the correction factors required in a spectrophotometric assay.
Methods and Results Eight- to 10-week-old C57BL6/J mice received either pH-neutral water (controls) or LPS (treatment) by intratracheal instillation, and acute lung injury was allowed to develop for 24 hours. Both control and treatment mice were further injected with Alb-EB via the jugular vein, at doses of either 20 or 30 mg/kg body weight, at 30, 60, 120, or 180 minutes before termination of the LPS treatment (24 hours total). At the end of exposure, formamide extracts of lungs were prepared, and the centrifuged supernatants were measured at 620 and 740 nm in a spectrophotometer. Lungs from control mice that were not injected with Alb-EB were similarly extracted and measured. The linear regression equation between absorbances at 740 nm (X) and 620 nm (Y) in control lung extracts of animals that did not get Alb-EB was considered to be the tissue-specific correction factor. The observed absorbances of the control and treatment samples at 620 and 740 nm were then normalized using this factor. This tissue-specific correction resulted in control samples read as positive integers, as opposed to negative integers when using a serum correction factor commonly used in the literature. We also determined that adjusting the duration of the conjugated dye in circulation is critical for maximizing the signal-to-noise ratio.
Conclusion The Evans Blue dye extravasation method to quantify barrier dysfunction can be improved in terms of repeatability and sensitivity by using tissue-specific correction factors and maximized signal-to-noise ratios, respectively.
Funding: NIH/NHLBI SCORR #P50 HL 73994.
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