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Antimicrobial Peptides pp 395–409Cite as

Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1548))

Abstract

Ease of access to the cornea makes antimicrobial peptides (AMPs) ideal candidates for topical drug application. However, before bringing them to the clinic, it is fundamental to evaluate in vitro: (1) the ability of AMPs to kill bacteria in the presence of human tears, by counting the number of surviving bacteria on agar plates; (2) the potential cytotoxicity of AMPs to mammalian cells by a colorimetric method based on the production of a colored formazan crystals by metabolically active cells; and (3) the ability of AMPs to neutralize the toxic effect of the bacterial cell wall component, lipopolysaccharide (LPS), by measuring the level of the pro-inflammatory cytokine, TNF-α, released from LPS-activated macrophages, using a sandwich enzyme-linked immunosorbent assay.

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Acknowledgments

This work was supported grants from Sapienza Università di Roma (MLM) and NIH grant EY13175 (AMM). Part of the content of this work is object of a US patent application No. 14/506,383.

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Authors and Affiliations

  1. Department of Biochemical Sciences, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy

    Floriana Cappiello, Bruno Casciaro & Maria Luisa Mangoni

  2. The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA

    Satya Sree Kolar, Hasna Baidouri & Alison M. McDermott

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  1. Floriana Cappiello
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  2. Bruno Casciaro
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  3. Satya Sree Kolar
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  4. Hasna Baidouri
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  5. Alison M. McDermott
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  6. Maria Luisa Mangoni
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Corresponding author

Correspondence to Maria Luisa Mangoni .

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Editors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Paul R. Hansen

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Cappiello, F., Casciaro, B., Kolar, S.S., Baidouri, H., McDermott, A.M., Mangoni, M.L. (2017). Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays. In: Hansen, P. (eds) Antimicrobial Peptides. Methods in Molecular Biology, vol 1548. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6737-7_29

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  • DOI: https://doi.org/10.1007/978-1-4939-6737-7_29

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6735-3

  • Online ISBN: 978-1-4939-6737-7

  • eBook Packages: Springer Protocols

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