NETs have attracted recent attention, but quantifying NETs in vivo has proven challenging. This protocol provides a desirable, highly sensitive, and valuable method for investigating the characteristics of NETs in clinical settings. This technique should extend to evaluate the severity of sepsis or septic ARDS, acute respiratory distress syndrome.
It is widely accepted that regulating dumps or damage-associated molecular patterns is a key to improving the clinical condition of sepsis. The greatest advantage of this method includes the accurate quantification of circulating NET remnants like myeloperoxidase-DNA and neutrophil elastase-DNA in a short time. It is advisable to spin the samples before assay and avoid repeated thawing.
It is important to follow the incubation times as given in the protocol. Begin by diluting freshly isolated polymorphonuclear neutrophils to 10, 000 cells per milliliter in phenol red-free RPMI 1640. Sieve them in 35-millimeter culture dishes.
To induce neutrophil extracellular traps or NETs, first stimulate the polymorphonuclear neutrophils with 25 nanomolar PMA. Then partially digest the extracellular traps by adding 0.6 micrograms per milliliter of DNase I, and incubate the mixture for 50 minutes at room temperature. Now, add five-millimolar EDTA to stop the DNase activity, and collect the medium containing the synthesized NETs.
Centrifuge to remove the cell debris. Collect the supernatants from four healthy controls. Mix store them at minus 80 degrees Celsius for further use.
Pipette 100 microliters of diluted anti-myeloperoxidase containing 0.05 micrograms of the antibody into an ELISA plate. Now, cover the plate with an adhesive plastic cover to prevent sample evaporation, and incubate the sample overnight at four degrees Celsius to allow the binding of the capture antibodies. The next day, discard the diluted antibody solution from the wells, and pipette 300 microliters of wash solution into each well.
Tap the plate dry on a paper towel to remove the excess PBS. Block each well of the plate with 200 microliters of the blocking buffer. Cover it with an adhesive plastic cover, and obstruct the wells by incubating it.
After discarding the blocking solution from the wells and washing the plate three to four times, tap it dry on a paper towel. Next, pipette 25 microliters of plasma into all wells except the blank. And dilute it with 75 microliters of PBS, making the final well volume 100 microliters.
Then add 100 microliters of PBS to the blank well. Mix the samples by placing the plate on a shaker for 10 seconds at 250 rpm at room temperature. Add two microliters of 100 full diluted DNase I to all wells, and place the sealed plate on the shaker for 10 seconds to thoroughly mix the samples.
Incubate for 15 minutes at room temperature. Add one microliter of 0.5 molar EDTA to each well to stop the DNase reaction. Then place the sealed plate on the shaker for 15 seconds to thoroughly mix the samples.
Finally, incubate the plate overnight at four degrees Celsius to allow the protein components of the NETs to attach to the capture antibodies. The next day, discard the solution from the wells. After multiple well washes, pipette 100 microliters of the diluted peroxidase-conjugated anti-DNA detection antibody into each well.
Incubate the plate for 1 1/2 hours, and then discard the solution in the wells. After washing the wells thrice, pipette 100 microliters of ABTS substrate solution into each well, and incubate the sealed plate on a shaker in the dark. Stop the reaction by adding 50 microliters of two-molar sulfuric acid.
Mix the well contents by carefully tapping the plate sides. Next, connect the microplate reader to the computer and launch the software application. From the status bar, create a new experiment and name it.
Then set the plate reading parameters by selecting Absorbance as the read type, Endpoint as the read mode, and two as the wavelengths. Next, set lambda one as 405 nanometers, lambda two as 490 nanometers. Now, select the Off status for both Automix and Blanking while keeping the AutoCalibration as On.Then select Read entire plate under Strips.
Finally, set the Column Wavelength Priority with Normal as the Carriage Speed, and then select Off under AutoRead. Place the plate well into the instrument drawer and close it. Click on Read so that the plate is read immediately.
Read the absorbance of each well at 405 nanometers, and perform automatic subtraction of the assay medium absorbance from all unknown samples. Reliable standard calibration curves were obtained for both MPO-DNA and NE-DNA when the absorbance values did not exceed 0.93 and 0.9, respectively. The highest OD was obtained when 0.6 micrograms per milliliter of DNase I was applied.
The inter-assay variability coefficients for the complexes in healthy controls were 1.871 and 0.987, respectively, while the COVID-19 patients showed variability coefficients of 2.532 and 2.010, respectively. The mean inter-assay variability coefficients for MPO-DNA and NE-DNA were 6.524 and 4.389, respectively. The capture antibodies'specificity to the MPO-DNA and NE-DNA complexes showed that the iso-type control antibodies reacted little to the complexes.
The percentage of both complexes was higher in the plasma from patients with COVID-19 relative to the healthy controls. DNase digestion time should be optimized, otherwise excessive digestion will reduce the absorbance.
