Our FRET probe and protocols enable the rapid and sensitive quantification of free and surface-bound elastase and cathespin G activity in sputum from patients with neutrophilic airway diseases. These methods, make it possible to explore different aspects of proteases pathophysiology. Plate reader measurements permit for large screenings.
Confocal microscopy visualizes enzyme activity with subcellular solution. While flow cytometry, enables single cell phenotyping and protease activity quantification in a personalized fashion. This technique can be expanded to different airway diseases such as cystic fibrosis, bronchiectasis or COPD.
And it can be adapted to different BioSamples such as blood or bronchial alveola lavash or mice samples. Before starting the sputum induction procedure, inhale 200 micrograms of the beta 2 receptor antagonists salbutamol. Afterwards, inhale hypertonic 6%saline solution for 15 minutes using a nebulizer.
Collect the expectorated sputum in a Petri dish. Separate mucus clumps from the saliva into a Petri dish with the help of a pipette tip. Weigh the mucus, then add four volume parts per weight of 10%Sputolysin and PBS to the sputum.
Incubate the mixture at room temperature on a rocking shaker for 15 minutes to dissolve the mucus. Quench the reaction by adding one milliliter of cold PBS for each milliliter of 10%Sputolysin. Pipette the mixture to obtain a homogeneous solution.
Filter the mixture through a 100 micron nylon cell strainer into a 50 milliliter tube. Repeat the filtration step through a 40 micron strainer, then centrifuge for 10 minutes at 300 times G at four degrees Celsius. Transfer the supernatant into a fresh tube and store it on ice.
Gently resuspend the cell pellet in 500 microliters of cold PBS and place it on ice. 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 the 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. For each measurement, resuspend 30, 000 sputum cells in a volume of 50 microliters of PBS in a 1.5 milliliter tube. Incubate the sputum cells with a specific inhibitor as a negative control and an appropriate enzyme as a positive control for 10 minutes at room temperature.
Add 50 microliters of PBS containing FRET reporter and a nuclear stain to each tube to get a final concentration of 2 micromolar. Incubate the mixture at room temperature for 10 to 20 minutes. Quench the reaction by adding 100 microliters of ice cold PBS and place the samples on ice.
Cytospin the mixture on microscopy slides. Then air dry and fix the cells with ice cold 10%methanol for 10 minutes. After fixation, air dry and mount the sample with an appropriate mounting medium.
Capture the images using a confocal microscope. Image at least 100 cells per condition for conclusive statistics. Resuspend 1 million cells in 100 microliters of PBS in a five milliliter FACS polystyrene round bottom tube, and place the tube on ice.
Add two microliters of FC block to each sample. Then incubate the samples for five minutes at room temperature. Add antibody each tube and incubate on ice in the dark for 30 minutes.
Wash the cells with two milliliters of cold PBS and centrifuge at 300 times G and four degrees Celsius. Finally, resuspend in 200 microliters of PBS. Divide the suspension into two tubes, 100 microliters each and add five microliters of cell viability stain.
Add appropriate specific NSP inhibitor to the negative control tube and incubate the sample for 10 minutes at room temperature in the dark. Add PBS to the sample and filter it through a 40 micron filter into a clean FACS tube. Add the reporter to the negative control sample and gently vortex.
Start acquiring cells incubated with this specific inhibitor and if necessary, slightly adjust the gates as well as the reporter PMTs voltages. To record changes in the donor to accept a ratio due to membrane bound protease activity, record 1000 neutrophils from each tube, every five to 10 minutes. Calculate the FRET ratio by dividing the donor by the acceptor channel values for the samples measured on the gated viable single neutrophils.
Images of neutrophils pre incubated with 100 micromolar Sivilestat or left untreated before reporter NEmo-2 addition are shown here. Nuclear signal was used to identify neutrophils by their characteristics segmented nuclei and the ROI was selected manually. The donor to accept a ratio of sputum neutrophils was plotted.
Each dot represents the mean of one ROI. Flow cytometry gating makes it possible to discriminate and study sputum neutrophils. The MFI distribution at zero and 10 minutes was observed after reporter addition.
The mean donor and acceptor MFI values for 1000 sputum neutrophils were calculated. The D/A ratio reached a plateau after an initial increase. The healthy donors sputum induction takes some practice before perfection.
Remember to relax and to breathe the aerosol for 10 to 15 minutes before expectoration. In addition, always keeps sputum cells on ice and process them as soon as possible after expectoration. We found that surface bound protease activity correlates with early lung damage in children and with severity of lung disease in adult CF patients.
Therefore together, these methods enable the exploration of proteases as early inflammation biomarkers in neutrophilic airway diseases.
