An In Vitro Assay for Neutrophil Serine Protease Activity Using a Fluorescent Reporter

Thaw enzymes on ice, and set up an enzyme standard curve as described in the text manuscript. In parallel to the standard preparation, dilute sputum samples in activation buffer. On the plate reader, set the excitation wavelength for the NE FRET probe NEmo-1 to 354 nanometers and the emission wavelength to 400 nanometers for donor and 490 nanometers for acceptor.

For the CG FRET probe sSAM, set the excitation wavelength to 405 nanometers, and emission to 485 nanometers for donor, and 580 nanometers for acceptor. Add 40 microliters of samples, standards, or blanks into the wells of a Black 96-well half-area plate and add the master mix.

Start the plate reader, and record the donor-to-acceptor ratio increase after every 60 to 90 seconds for at least 20 minutes or until the increase in the signal reaches a plateau. Export the data and calculate the donor-to-acceptor ratio by dividing the donor RFU by the acceptor RFU for each time point and sample. Then, calculate the donor-to-acceptor ratio mean and standard deviation. Determine the slope within the linear growth of the donor-to-acceptor ratio change.