This protocol demonstrates the heterogeneity in composition, structure, and morphology of neutrophil extracellular traps, depending on their inducing stimulus under comparable in vitro conditions in human neutrophils from healthy individuals. High neutrophil purity and viability are obtained. That allows to compare the concentration of DNA, LL-37, and enzyme activity of myeloperoxidase, elastase, and cathepsin G, released by neutrophil extracellular traps.
This protocol allowed to analyze the effect of soluble factors or cellular contact or possible therapies or mechanisms for closing on the production of NETs in autoimmune disease. These methods can aid in the investigation of autoimmune disease. Due to the uncontrolled cell reproduction of NETs and duration of the align their components, which are considered biomarkers of the disease.
Fluorescence microscopy allows to capture image of NETs showing the dispersion of DNA in association with protein such as LL37, which co-localized with microorganisms helping to understand how they are appearing. To begin, perform vena puncture to collect 10 milliliters of peripheral blood in tubes containing dipotassium EDTA as anticoagulant. Then perform standard blood biometry and C-reactive protein test to rule out infection or inflammation to ensure the quality of the sample.
Centrifuge the peripheral blood sample to remove the platelet-rich plasma, followed by a second centrifugation and discard the remaining plasma. Dilute the resulting erythrocyte and leukocyte package in one-to-one volume ratio with one 1X DPBS. Then, in a sterile 10 milliliter glass tube, first deposit one milliliter of 1.08 grams per milliliter density solution followed by one milliliter of 1.079 grams per milliliter density solution.
Then, add four milliliters of the diluted erythrocyte and leukocyte package by pouring over the walls. Centrifuge without acceleration or deceleration to avoid disturbing the gradient. Aspirate the phase corresponding to granulocytes and transfer to another sterile 10 milliliter glass tube.
Wash with four milliliters of 1X DPBS at 300 G for 10 minutes at four degrees Celsius and discard the supernatant. To remove the remaining erythrocytes, treat the cells with osmotic shock by adding four milliliters of 0.2%saline solution. Incubate for two minutes at four degrees Celsius and centrifuge.
Discard the supernatant and add four milliliters of 0.65%saline solution. Incubate for five minutes at four degrees Celsius to restore membrane integrity and repeat centrifugation. Remove the supernatant and re-suspend the cells in four milliliters of 1X DPBS to remove cellular debris.
Again, centrifuge and re-suspend the cell pellet in two milliliters of cold HBSS buffer. To perform a trypan blue exclusion test, dilute five microliters of the cell suspension in 20 microliters of 0.4%trypan blue. Count the cells in a new bower chamber and determine cell viability using an exclusion test.
Then, mount 5 microliters of the cell suspension on a slide and stain with Wright's stain for 15 seconds. Immediately fix the sample, wash with distilled water, and observe the morphology under an optical microscope. Next, add one times 10 to the fifth cells to flow cytometry tubes and stain with one microliter of 7AAD in 100 microliters of FACS buffer for 15 minutes at four degrees Celsius in the dark.
Wash with 500 microliters of FACS buffer at 300 G for 10 minutes. Fix the cells with 500 microliters of 2%paraformaldehyde and store at four degrees Celsius until flow cytometry analysis. For a dead cell control, fix the cells with 200 microliters of 4%paraformaldehyde for 30 minutes and wash with 500 microliters of 1XPBS at 300 G for 10 minutes at four degrees Celsius.
Discard the supernatant and add 200 microliters of 0.1%Triton X-100. Incubate for one hour at four degrees Celsius. Wash with 500 microliters of 1X PBS and stain with 7AAD.
Analyze the captured data in the flow cytometer software. Load the files and double click on the unstained neutrophil file. Choose forward scatter or FSC on the X-axis and side scatter or SSC on the Y-axis to display the cell population corresponding to neutrophils.
Then select the channel B3-A:PerCP vio 700-A on the X-axis to de-limit the autofluorescence of the cells and choose histogram on the Y-axis. Then, open the stained neutrophil file. Choose FSC on the X-axis and SSC on the Y-axis to display the cell population corresponding to neutrophils.
Again, select the channel B3-A:PerCP vio 700-A to delimit the population of neutrophils positive for the dye 7AAD. Generate the final histogram by right-clicking on the window and selecting copy to layout editor. Add bacterial or fungal pseudohyphae in 1.5 milliliter micro tubes.
Add 200 microliters, so five micromolar CFSC in 1X PBS. Mix and incubate at 37 degrees Celsius for 10 minutes in the dark. Stop the reaction by adding 500 microliters of decomplemented plasma and centrifuge.
Discard the supernatant and wash the pellet with one milliliter of 1XPBS with centrifugation. Then re-suspend the microorganisms in 250 microliters of 1X PBS. Prepare 50 microliter aliquots in 1.5 milliliter microtubes with two times 10 to the seventh bacteria or two times 10 to the fifth pseudohyphae for NET induction.
Place 10 by 10 millimeters sterile glass cover slips in a 24-well plate. Cover with 10 microliters of 0.001%poly l-lysine for one hour at room temperature and wash twice with 100 microliters of 1X PBS. Air dry and irradiate with UV light for 15 minutes.
Next, replace HBSS solution of the neutrophil suspension prepared earlier with RPMI 1640 medium supplemented with 10%heat decomplemented autologous plasma. Add 350 microliters of this cell suspension to the 24-well plate for a final concentration of two times 10 to the fifth neutrophils per well. Incubate the plate for 20 minutes at 37 degrees Celsius with 5%carbon dioxide to allow the cells to adhere to the bottom of the wells.
To induce net formation, add the bacteria stimuli MOI100 and pseudohyphae at MOI1. Also add the biochemical stimuli and prepare a control without a stimulus by adding 50 microliters of HBSS. Obtain a final volume of 400 microliters per well and mix on a plate shaker at 140 RPM for 30 seconds.
Then incubate for four hours at 37 degrees Celsius and 5%carbon dioxide. After net induction, remove the supernatant from the wells by pipetting carefully and fix the cells with 300 microliters of 4%paraformaldehyde for 30 minutes. Wash the cells with 200 microliters of 1X PBS without centrifuging and add 200 microliters of blocking buffer for 30 minutes.
For LL37 stain, permeabilize the cells with 200 microliters of 0.2%Triton X-100 in 1X PBS for 10 minutes and wash with 1X PBS twice. Mount the cover slips on glass slides. DNA stain the cells with two microliters of DAPI and store at minus 20 degree Celsius until analysis by confocal fluorescence microscopy.
The dynamic cellular phases were visualized after double density gradient purification. The typical neutrophil morphology was confirmed by right staining. The cells also showed optimal viability observed by trypan blue exclusion without membrane disruption.
Analysis of SSC versus FSC by flow cytometry confirmed a population corresponding to PMN with high cell purity. PMN dot plots for unstained cells versus 7AAD stain cells and their respective histograms indicated a high cell viability in the freshly isolated neutrophils compared to the permeabilized control cells. After stimulating the neutrophils with chemical and microbial stimulants, NETs were visualized by fluorescence microscopy using DNA DPI and anti-LL37.
The microorganisms were pre-stained with CFSE. PMA-induced suicidal net formation indicated by co-localization with LL37. Whereas NETs formed with HOCI showed minor DNA dispersion in the extracellular space that corresponded to vital NET formation.
NET induced by fungal pseudohyphae showed low concentrations of DNA released into the extracellular space with filamentous structures characteristic of the vital NET formation. S.aureus showed vital NET formation whereas P.aeruginosa induced the release of large concentrations of DNA with a cloudy morphology that co-localized with LL37 indicating suicide NET formation. Performing double density gradient methodology allows us to obtain a high purity and viability of neutrophils.
And we elevate the washings, we avoid damaging the structure of them. Following this method we can analyze the effect of substances or cells in NET production as well as if they are involved in some mechanism or their use as therapies in autoimmune disease.
