Quantifying Bacterial Infection-Induced Amyloids Using Thioflavin T Fluorescence

Abstract

Source: Balczon, R., et al. Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa. J. Vis. Exp. (2018)

This video demonstrates the protocol to quantify amyloids in culture supernatants from pulmonary endothelial cells infected with virulent bacteria, using thioflavin T, a fluorescent dye that binds to amyloid beta-sheet structures. An increase in fluorescence compared to baseline indicates the presence of amyloids in the culture supernatant.

Protocol

1. Analysis of Cytotoxic Supernatants

1. To do a thioflavin T (ThT) assay, use ThT fluorescence to quantify amyloids in supernatants. For these measurements, use filter-sterilized supernatants.
   1. Prepare a stock solution (50x) of ThT by suspending 8 mg ThT in 10 mL phosphate-buffered saline (PBS). After mixing, filter the solution through a 0.22 µm filter to remove particulates.
   2. For measurements, add 20 µL of ThT stock to 1 mL of PBS in a 1 mL spectrophotometer cuvette. Place the diluted sample into a spectrofluorometer.
   3. Measure the baseline fluorescence emission using 425 nm excitation and scanning t....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thioflavin T	Sigma Aldrich	T3516
PBS	Gibco	10010-023
0.22 micron syringe filters	Millipore	SLGP033RS