Quantifying Bacterial Infection-Induced Amyloids Using Thioflavin T Fluorescence

Samenvatting

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 the fluorescence emission from 450-575 nm in 2 nm increments.
   4. Perform a time-lapse scan using 425 nm excitation and 482 nm emission, with data acquired every 0.2 s for 60 s.
   5. The initial 20 s of the time-lapse scan measures fluorescence in the blank cuvette. At 20 s, pause the scan and add 10 µL of the filter-sterilized supernatant to the cuvette. Mix the cuvette by inversion and then place it back into the spectrofluorometer.
   6. Resume the time-based scan, acquiring the final 40 s of data.
   7. Upon completion of the time-lapse scan, perform a final fluorescence emission spectrum scan using identical settings, as detailed in step 1.3.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Thioflavin T	Sigma Aldrich	T3516
PBS	Gibco	10010-023
0.22 micron syringe filters	Millipore	SLGP033RS