Quantifying Bacterial Infection-Induced Amyloids Using Thioflavin T Fluorescence

Take a cuvette containing a working solution of thioflavin T, an amyloid-specific fluorescent dye, dissolved in phosphate-buffered saline.

Place the cuvette in a spectrofluorometer and record the baseline fluorescence signal.

Pause the scan and remove the cuvette.

Add the filter-sterilized culture supernatant collected from pulmonary endothelial cells infected with virulent Pseudomonas bacteria.

The supernatant contains amyloid aggregates, which are misfolded, beta-sheet-rich proteins.

Gently mix the contents by inversion to ensure uniform distribution.

Reload the cuvette in the spectrofluorometer and resume the scan to acquire a time-based fluorescence signal.

Thioflavin T binds specifically to beta-sheet-rich amyloids, which leads to enhanced fluorescence.

Perform a fluorescence scan using the same settings.

An increase in fluorescence relative to the baseline indicates the presence and relative abundance of infection-induced amyloids in the culture supernatant.

To quantify the amyloids within the supernatant, add 20 microliters of freshly prepared and filtered 50X thioflavin T stock solution to one milliliter of PBS in a one milliliter spectrophotometer cuvette, and load the diluted sampl