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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 Methods in Molecular Biology book series (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.

Key words

  • Colony counts
  • Tear collection
  • Colorimetric MTT assay
  • Sandwich immunosorbent assay
  • LPS detoxification

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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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Correspondence to Maria Luisa Mangoni .

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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

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