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

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.

Keywords: Colony counts; Colorimetric MTT assay; LPS detoxification; Sandwich immunosorbent assay; Tear collection.

Fig. 1

Collection of basal tears. Obtain tears by inserting a 5 μl glass capillary tube (a) into the fornix (indicated by the arrow), in a perpendicular orientation to the ocular surface (b)

Fig. 2

Example of viability assay using the colored indicator MTT. The intensity of the purple dye (formazan) is proportional to the number of viable cells. The transparent yellow color in the B11 well corresponds to MTT solution without cells

Fig. 3

Representation of TNF-α standard dilutions and their loading in a 96-well plate. Put 500 μl of Assay Diluent 1× in seven Eppendorf tubes, from A to G, and perform twofold serial dilutions by transferring 500 μl from the top standard (2000 pg/ml) to tube A, then from A to B, and so on until G. Transfer 100 μl of diluted standards in the corresponding wells (in triplicate) and load Assay Diluent 1× only in three wells H

Fig. 4

Schematic representation of a sandwich enzyme-linked immunosorbent assay (ELISA). The plate is coated with a suitable capture antibody (1). Then the sample is added and the antigen present is bound to the capture antibody (2). A suitable biotin-labeled detection antibody (BDAb) which binds to the antigen is added (3). Avidin-HRP binds BDAb (4). Finally, TMB substrate is added and converted to a detectable form

Fig. 5

Representation of a Burker and a Neubauer chamber. Left side: using a micropipette tip (indicated by the arrow), inject 10 μl of cell suspension under the cover glass (previously put over the chamber). It is recommended to count both sides (1 and 2) of the chamber. Right side: typical grids of Burker and Neubauer chambers, showing squares, each of which is 1 mm long and contains 16 smaller squares