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 sample onto a spectrofluorometer.

Measure the baseline fluorescence emission using a 425 nanometer excitation. Scanning the fluorescence emission from 450 to 575 nanometers in two nanometer increments. Then set the instrument to perform a time lapse scan using a 425 nanometer excitation and 482 nanometer emission with data acquisition every 0.2 seconds for 60 seconds.

Pause the scan after 20 seconds and add 10 microliters of filter sterilized supernatant to the cuvette. After mixing by inversion, reload the cuvette onto the spectrofluorometer and resume the time based scan for the final 40 seconds. Upon completion of the time lapse, perform a final fluorescence emissions spectrum scan using the original scan settings.