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Originally published In Press as doi:10.1194/jlr.D800029-JLR200 on July 18, 2008

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Journal of Lipid Research, Vol. 49, 2479-2488, November 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

High-throughput evaluation of pulmonary surfactant adsorption and surface film formation

Andrea Ravasio1,*, Antonio Cruz{dagger}, Jesús Pérez-Gil{dagger} and Thomas Haller*

* Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria
{dagger} Departamento Bioquímica, Facultad de Biología, Universidad Complutense, Madrid, Spain

Published, JLR Papers in Press, July 18, 2008.

This research has been supported by grants from FWF Austrian Science Fund (P17501) to T.H. and the Spanish Ministry of Science (BIO2006-03130, CONSOLIDER-INGENIO 2010 CSD2007-00010) and Community of Madrid (S0505/MAT/0283) to J.P.-G. and A.C. Collaboration between Austrian and Spanish teams has been facilitated in the context of the Marie Curie Network PULMO-NET (RTN-512229).

1 To whom correspondence should be addressed. e-mail: and.ravasio{at}gmail.com

The assessment of new therapeutic strategies to cure surfactant-associated lung disorders would greatly benefit from assay systems allowing routine evaluations of surfactant functions. We present a method to measure surfactant adsorption kinetics into interfacial air-liquid interfaces based on fluorescence microplate readers. The principle of measurement is simple, robust, and reproducible: Wells of a microtiter plate contain an aqueous solution of a light-absorbing agent. Fluorescence is excited and collected from the top of the wells so that fluorescently labeled surfactant injected into the bulk can be detected only once adsorbed into the air-liquid interface. Mass transfer from the bulk to the interface is achieved by orbital shaking implemented in the plate reader instrument. The method has been tested and validated by using phospholipids or surfactants of different origins, by using albumin as surfactant inhibitor, and by comparison of results with Wilhelmy balance measurements. The method is suited for implementation in high-throughput screening routines for conditions affecting, or improving, surfactant film formation. In contrast to surface tension measurements, our method gives a direct readout of the amount of surfactant adsorbing into the interface, including the functionally important amount of material firmly associating with the interfacial film.

Supplementary key words air-liquid interface • fluorescence • lamellar bodies • lung • microplate • surface tension

Abbreviations: AT II, alveolar type II; BB, Brilliant Black; CBS, captive bubble surfactometer; LBP, lamellar body-like particle; LTG, LysoTracker Green; NS, native surfactant; PBS, pulsating bubble surfactometer; RFU-bg, background-corrected relative fluorescence units


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