The overall goal of this procedure is to quantitate neutrophil extracellular traps, or NETs, released from human neutrophils on a high throughput scale. The methods presented here can help answer key questions in the fields of inflammation and altering inflammatory diseases through the study of the mechanism and regulation of in vitro neutrophil extracellular trap formation in human neutrophils. The implications of this technique involves identification of a central NET mediating signaling pathways and NET inhibitory or NET stimulating molecules.
To isolate neutrophils from blood, aliquot 20 milliliters of human blood into 250 milliliter conical tubes. Add 10 milliliters of 6%dextran, then gently mix and allow the solutions to sit at room temperature for 20 minutes. Carefully transfer the leukocyte rich upper phase into clean 50 milliliter conical tubes, and use sterile PBS to fill the tube to the top.
Then centrifuge the sample at 400 times G for ten minutes and use 15 milliliters of sterile PBS to resuspend the pellet. Repeat this wash step one more time. Next, prepare five step percoll gradients of 85, 80, 75, 70 and 65 percent in 215 milliliter conical tubes and layer two milliliters of the leukocyte suspension on top of each gradient.
Then, centrifuge the gradients at 800 times G for 30 minutes with the break off. After centrifugation, collect the cells in the layers at the 75-80%and 70-75%interfaces. Use PBS to wash the cells twice, and then use a hemocytometer to count the cells.
To measure the kinetics of extracellular DNA release, prepare a suspension of neutrophils in assay medium at a concentration of two times ten to the six cells per milliliter. Add five micromoles per liter of SYTOX orange, which is a membrane-impermeable DNA binding dye and gently mix. Aliquot human neutrophils into sterile 96-well black transparent bottom microplates ensuring that the assay medium covers the entire bottom of the well.
If not, gently tap the plate on the side. Warm the plate at 37 degrees celsius for 10 minutes. In the mean time, use 37 degrees celsius assay medium to prepare solutions containing stimuli at double their final concentrations to be used.
As a positive NET control, prepare 100 nanomolar phorbol myristate acetate, or PMA, to treat human neutrophils for four hours to trigger maximal NET release. Next, prepare the microplate fluorometer for measurement. Stimulate neutrophils by adding 50 microliters of stimulus solution to 50 microliters of neutrophils suspensions.
Add a final concentration of 0.5 milligrams per milliliter of saponin to one well, to measure the maximal DNA release signal. Place the plate in the reader and measure changes in fluorescence at two minute intervals for four hours without shaking. To analyze the data, calculate normalized increases in fluorescence over time by subtracting the baseline fluorescence from end point values for all wells including saponin.
The rise in saponin fluorescence should be the highest signal and is referred to as the maximal DNA release. Average the results of replicates and present them as percent of maximal DNA release. To quantitate NET formation using MPO-DNA and HNE-DNA ELISA assays, add anti-MPO or anti-HNE capture antibodies to 96-well high binding capacity ELISA plates, and incubate them overnight to coat.
The next morning use 200 microliters per well of PBS to wash the ELISA plates three times. Then use 200 microliters of 5%bovine serum albumin, or BSA, and 0.1%human serum albumin, or HSA, to block the wells at room temperature for two hours. In the mean time, seed neutrophils in 100 microliters of assay medium on a 96-well plate at a density of 100, 000 cells per well and induce NET formation after addition of stimuli by incubating at 37 degrees celsius for four hours.
Next, perform a limited DNase digestion by adding two units per milliliter of DNAse to the neutrophils supernatants then mix well and incubate at room temperature for 15 minutes. Then, stop the reaction by adding 15 microliters of 25 millimolar EGTA and mix well. Collect the supernatants and clean microfuge tubes and spin the samples to get rid of cell debris at 300 times G and room temperature for five minutes.
Collecting NETs without limited digestion results in inconsistent data. The limited DNA digestion step is critical to obtain reproducible results. Therefore, the concentration of DNase I enzyme and the length of digestion has been optimized.
Transfer the supernatant to clean microfuge tubes and keep on ice. Then, using an aliquot of previously prepared NET-standard, prepare a one to two dilution in PBS and EGTA. Now, use PBS with EGTA to dilute DNase I digested samples twenty fold and aliquot them on ELISA plates coated with capture antibodies.
Incubate the plates over night at four degrees celsius. Before using PBS to wash the plates three times, apply 100 microliters per well of a detection antibody solution such as horseradish peroxidase conjugated anti DNA antibody, and incubate in the dark for one hour. After four washes with PBS, add 100 microliters per well of TMB peroxidase substrate, and incubate for 30 minutes.
Blue coloration indicates peroxidase activity and the presence of NETs. To stop the reaction, add 100 microliters of one molar HCL. The blue solutions will turn yellow.
Using a microplate photometer, read the absorbance at 450 nanometers. To compare the amount of MPO-DNA or HNE-DNA complexes measured compared to the NET-standard, subtract background optimal density from all samples and plot the absorbance values of the diluted standard samples against their relative NET content. Using the non-saturated range of this standard curve, establish a trend line using the best exponential fit from the software used.
Insert the measured OD values of the unknowns into the trend line equation that will give the amount of NETs in the unknown sample as percent of the NET-standard. Finally, calculate the averages of replicates and present the final data as the amount of MPO-DNA or HNE-DNA complexes. Shown here is representative data from a fluorescence based DNA release assay of human neutrophils left un-stimulated or activated with PMA or P-aeruginosa.
P-aeruginosa triggers NET formation in human PMNs. This graph illustrates that the averages of increase in fluorescence of replicate wells are calculated, normalized on maximal fluoresence and are expressed as DNA release. These fluorescence images represent neutrophils left untreated or stimulated with PMA.
Shown here are immunofluorescence images of nonstimulated, PMA, and PA01 activated human neutrophils from an MPO-DNA and HNE-DNA Elisa assay. Extracellular DNA shows typical NET morphology and colocalizes with MPO and citrullinated histone H4, a marker of NET formation. This experiment shows MPO-DNA and HNE-DNA release in human neutrophils using the same assay.
The NET-standard on average contains 19.5 micrograms per milliliter of extracellular DNA, 399 nanograms per milliliter of MPO, and 103.4 nano-grams per milliliter of HNE. Based on the molecular weights of MPO and HNE, and that one microgram of DNA contains 9.91 times 10 to the 14 nucleotides, the NET-standard contains 109 HNE molecules and 147 MPO molecules per 1, 000 nucleotides. Finally, two fold serial dilution experiments of the NET-standard reveal that the dynamic ranges of both the MPO-DNA and HNE-DNA ELISA assays are between 19 and 609 nanograms per milliliter of DNA.
Once mastered, the SYTOX orange based acid can be easily and quickly assembled in one hour, leading to results the next day. The NET quantitating assay are also easy to perform and will result in data the following day if performed properly. After watching this video, you should have a good understanding of how to quantitate naturalase in human neutrophils in a high throughput manner.
