In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

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Stem Cells
Sep 2016


In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange.

Keywords: NETs (neutrophil extracellular traps) (NETs(中性粒细胞胞外诱捕网)), Neutrophil (中性粒细胞), Sytox orange (Sytox橙色), DNA/Histone H1 (DNA/组蛋白H1)


Neutrophils constitute the largest, evolutionary conserved fraction of circulating leukocytes. They set up the first defence line against pathogens by various mechanisms including the formation of neutrophil extracellular traps (NETs). During this process, activated neutrophils expel chromatin fibers from the nucleus. Invading pathogens are then trapped within the network of chromatin and killed by highly concentrated, NET-entangled antimicrobial proteins, such as myeloperoxidase (MPO) and elastase (Brinkmann et al., 2004). However, NETs are a double-edged sword; the unrestrained NET formation from over-activated neutrophils can also contribute to severe tissue damage, for instance by the cytotoxic effect of histone components of NETs (Saffarzadeh et al., 2012). One example of a pathological condition in which neutrophils are over-activated and have enhanced capacity to form NETs is systemic lupus erythematosus. The levels of antibodies against double-stranded DNA as well as other components of NETs are elevated in sera of lupus patients (Knight and Kaplan, 2012; Yu and Su, 2013). Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which cause endothelial cell damage, a critical step in the pathogenesis of lupus and other neutrophil over-activation syndromes (Villanueva et al., 2011).

Different methods have been used for NET detection and quantification, including immunocytochemistry (Brinkmann et al., 2010), fluorescent dyes, flow cytometry, and ELISA. Immunocytochemistry with DAPI and DNA/histone was the best method for NET qualification and quantification, since decondensation of the nucleus indicates NET formation. Picogreen dye is a sensitive method to quantify NET-DNA concentration (Saffarzadeh et al., 2012; Tanaka et al., 2015), while Sytox orange is a fast and easy method for NET quantification. Flow cytometric analysis by measuring the signal for labeled-DNA/histone antibody, or myeloperoxidase-DNA based ELISA are useful methods for detection and quantification of NETs in pathological samples such as serum or peritoneal fluid of patients (Caudrillier et al., 2012).

In this protocol, we provide the details for the demonstration of NET formation by fluorescent immunostaining for chromatin fiber with anti-DNA/histone H1 antibody, which has a very high affinity for decondensed chromatin in NETs in comparison to DAPI or Hoechst (Saffarzadeh et al., 2012). Furthermore, we describe a quick method for NET quantification with Sytox orange, a non-cell-permeable DNA specific dye staining extracellular DNA content (Williams et al., 1999; Yost et al., 2009), by fluorescence intensity measured by a microplate reader. This protocol has been applied successfully in our recent studies, whereby we show that antibody- or complement-induced phagocytosis triggers rapid NET formation (Saffarzadeh et al., 2014), and more importantly, mesenchymal stem cells suppress NET formation from over-activated neutrophils (Jiang et al., 2016).

Materials and Reagents

  1. Pipette tips (Greiner Bio One International, Ultratip)
  2. Tube 50 ml (SARSTEDT, catalog number: 62.547.254 )
  3. Syringe 10 ml (B. Braun medical, catalog number: 4606108V-02 )
  4. Needle 26 G x ½” (B. Braun medical, catalog number: 4665457-02 )
  5. Pre-Separation filters (30 µm) (Miltenyi Biotec, catalog number: 130-041-407 )
  6. Millicell EZ Slide 8-well glass (EMD Millipore, catalog number: PEZGS0816 )
  7. 96-well, black, flat bottom plate, sterile, with lid (Corning, catalog number: 3916 )
  8. C57BL/6J mice at preferred age of 8-12 weeks, both male and female are suitable for this protocol (THE JACKSON LABORATORY, catalog number: 000664 )
  9. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  10. 0.5 M EDTA, sterile (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575020 )
  11. Histopaque-1077 (Sigma-Aldrich, catalog number: 10771 )
  12. Histopaque-1119 (Sigma-Aldrich, catalog number: 11191 )
  13. Optional: FITC-conjugated mouse anti-human CD15 antibody (Clone VIMC6) (Miltenyi Biotec, catalog number: 130-081-101 )
  14. Optional: FITC-conjugated mouse IgM isotype control (Miltenyi Biotec, catalog number: 130-093-178 )
  15. Optional: MACSxpress neutrophil isolation kit, human (Miltenyi Biotec, catalog number: 130-104-434 )
  16. Optional: MACSxpress erythrocyte depletion kit, human (Miltenyi Biotec, catalog number: 130-098-196 )
  17. Mouse neutrophil isolation kit (Miltenyi Biotec, catalog number: 130-097-658 )
  18. Optional: Propidium iodide (PI) staining solution (BD, BD Biosciences, catalog number: 556463 )
  19. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
    Note: Dissolve in DMSO to make 1 mg/ml stock, and store the aliquots at -20 °C.
  20. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  21. 16% (w/v) paraformaldehyde (PFA), methanol-free (Thermo Fisher Scientific, catalog number: 28908 )
  22. Purified mouse anti-DNA/Histone H1 antibody (EMD Millipore, catalog number: MAB3864 )
    Note: Make aliquots and store at -20 °C.
  23. 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  24. Alexa Fluor 555-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-21422 )
  25. Optional: APC-conjugated rat anti-mouse Ly6G (Gr-1) antibody (Clone RB6-8C5) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 17-5931-82 )
  26. Optional: APC-conjugated rat IgG2b Isotype control (Clone eB149/10H5) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 17-4031-82 )
  27. Optional: purified rat anti-mouse Ly6G antibody (Clone RB6-8C5) (Abcam, catalog number: ab25377 )
  28. Optional: purified rabbit anti-human CD15 antibody (Clone SP159) (Novus Biologicals, catalog number: NBP2-21754 )
  29. Purified goat anti-human/mouse myeloperoxidase/MPO antibody (R&D Systems, catalog number: AF3667 )
  30. Purified mouse IgG2a isotype control (Clone C1.18.4) (BD, BD Biosciences, catalog number: 550339 )
  31. Purified rabbit anti-human/mouse neutrophil elastase antibody (Abcam, catalog number: ab68672 )
  32. Sytox orange nucleic acid stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11368 )
  33. Fetal bovine serum (FBS) (Biochrom, catalog number: S 0615 )
  34. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
  35. RPMI 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21875034 )
  36. GlutaMAX (Thermo Fisher Scientific, catalog number: 35050038 )
  37. MEM non-essential amino acids (NEAA) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140035 )
  38. Penicillin/streptomycin (Biochrom, catalog number: A 2213 )
  39. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  40. Goat serum (Sigma-Aldrich, catalog number: G9023 )
  41. Fluorescence mounting medium (Agilent Technologies, DAKO, catalog number: S302380-2 )
  42. FACS buffer (can be kept at 4 °C for 2 weeks) (see Recipe 1)
  43. R1 medium (prepared media can be kept at 4 °C for 2 weeks) (see Recipe 2)
  44. Blocking buffer (prepare fresh) (see Recipe 3)
  45. Antibody diluent (prepare fresh) (see Recipe 4)


  1. Pipettes
  2. Centrifuge
  3. Optional: MACSxpress Separator (Miltenyi Biotec, catalog number: 130-098-308 )
  4. Optional: MACSmixTM Tube Rotator (Miltenyi Biotec, catalog number: 130-090-753 )
  5. Humidified cell culture incubator set to 37 °C and 5% CO2
  6. Orbital shaker, such as Heidolph Unimax 1010 (Heidolph Instruments, model: Unimax 1010 , catalog number: 543-12310-00)
  7. Fluorescent microscope, such as Zeiss Axiophot microscope with an AxioCam digital color camera and AxioVision software v4.7 (Carl Zeiss, model: Axiophot )
  8. AxioCam digital color camera
  9. Microplate reader that can measure the absorbance and emission of Sytox orange at 547 nm and 570 nm, respectively, such as Mithras LB940 (BERTHOLD TECHNOLOGIES, model: Mithras LB 940 )
  10. Optional: Flow cytometer (to check the purity of isolated neutrophils) FACS Canto II (BD, BD Biosciences, model: BD FACSCANTO II ) with FACSDiva software


  1. AxioVision software
  2. Optional: FACSDiva software


  1. Neutrophil isolation
    1. Isolate human neutrophils from fresh peripheral blood using gradient centrifugation (Figures 1A and 1B)
      1. Dilute peripheral blood 1:2 with PBS containing 2 mM EDTA.
      2. In each 50 ml tube, carefully and sequentially lay 10 ml Histopaque-1119, 10 ml Histopaque-1077 and 25 ml diluted blood.
        Note: Histopaque should be brought to room temperature prior to use.
      3. Centrifugation at 700 x g (Note: Without break.) at room temperature for 30 min.
      4. Transfer the layer at the interface of Histopaque-1077 and Histopaque-1119 to a new 50 ml tube (the usual volume ranges from 2-3 ml), top up to 50 ml with PBS and centrifuge at 300 x g at room temperature for 10 min.
      5. Wash again with 30 ml PBS.
        Note: The expected average yield of neutrophils per ml of peripheral blood from healthy donor ranges from 2-5 million cells.

        Figure 1. Neutrophil isolation. A. Human neutrophils were isolated from fresh peripheral blood of healthy donors by gradient centrifugation (step A1). The neutrophil layer at the interface of H1077 and H1119 were collected. PBMC, peripheral blood mononuclear cells; PMN, polymorphonuclear leukocytes; RBC, red blood cells. B. The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed using a human CD15 antibody within the PI negative gate. C. Human neutrophils were isolated from buffy coat with the MACSxpress method (step A2). The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed with human CD15 antibody within the PI negative gate. D. Murine bone marrow neutrophils were isolated by magnetic sorting using a negative depletion method (step A3). The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed with a mouse Ly6G (Gr-1) antibody within the PI negative gate. Gray histograms: PI dye or CD15 antibody or Gr-1 antibody; open histogram: unstained control or isotype antibody.

    2. Alternative: Isolate human neutrophils with MACSxpress neutrophil isolation kit (Figure 1C)
      1. Mix 5 ml of buffy coat (Note: less than 12 h after collection.) with 1 ml reconstituted buffer A (kit component), 1 ml buffer B and 100 µl reconstituted MACSxpress erythrocyte depletion reagent.
      2. Incubate sample for 5 min at room temperature using the MACSmix Tube Rotator at 12 rpm.
      3. Place the open tube in the magnetic field of the MACSxpress Separator for 10 min, then carefully collect the supernatant containing neutrophils into a new tube.
    3. Isolation of murine bone marrow neutrophils (Figure 1D)
      1. Dissect femur bones of C57BL/6J mice, and flush out the bone marrow with 10 ml PBS containing 2 mM EDTA by using a 10 ml syringe and 26 G needle aseptically.
      2. Filter the suspension with a 30 µm cell strainer.
      3. Isolate neutrophils by immunomagnetic separation (MACS) using the negative depletion method with mouse neutrophil isolation kit (Miltenyi), following the manufacturer’s instructions.
        Note: The expected average yield of neutrophils from bone marrow of two femur bones of one C57BL/6J mouse at age of 8-12 weeks ranges from 10-15 million cells.
    4. Optional: Check purity and viability of isolated neutrophils
      1. Resuspend 2 x 105 isolated human neutrophils in 100 µl FACS buffer (see Recipe 1) containing 5 µl of anti-human CD15-FITC antibody (or anti-mouse Gr1-APC antibody for mouse neutrophils). Another 2 x 105 cells in 100 µl FACS buffer containing 5 µl of mouse IgM-FITC isotype control (or rat IgG2b-APC isotype control for mouse neutrophils). Incubate at room temperature in the dark for 30 min.
      2. Add 100 µl FACS buffer, mix gently, and centrifuge to collect cell pellet with a benchtop centrifuge at 3,000 RPM (870 x g with 87 mm rotor) at room temperature, remove supernatant.
      3. Resuspend pellet in 300 µl FACS buffer, add 3 µl Propidium iodide (PI) staining solution, mix quickly.
      4. Proceed to flow cytometric analysis for CD15+PI- cells (human), or Gr-1hiPI- cells (mouse).

  2. NET demonstration by immunostaining
    Note: If the aim is to study the interaction of specific adherent cells with neutrophils and NET formation, seed the cells of interest one day prior to neutrophil isolation into 8-well glass chamber slide (Millicell EZ Slide 8-well glass) in 300 µl medium per well and culture overnight. The seeding cell number depends on the ratio of the target cells to neutrophils. Make duplicate wells for each condition. Remove culture medium before adding neutrophils.
    1. Induction of NET formation
      1. Add freshly isolated neutrophils into 8-well glass chamber slide at a density of 2 x 105 neutrophils in 300 µl R1 medium (see Recipe 2) per well. Make duplicate wells for each condition.
        Note: Resuspend neutrophils in R1 medium at 6.67 x 105 cells/ml, then add 300 µl per well.
      2. Add the treatment of interest (e.g., potential chemical activator/inhibitor of NET, bacteria, interacting cells, etc.)
        Note: Incubation time needs to be optimized individually, more details see Note 1.
      3. Dissolve PMA in DMSO to make 1 mg/ml stock solution. To the positive control wells, and conditions that NET formation are desired, add PMA at a final concentration of 100 ng/ml to activate neutrophils (i.e., dilute PMA stock 1:100 in R1 medium, then add 3 µl of diluted PMA to each well). (see Note 2)
      4. Dilute DMSO 1:100 in R1 medium, add 3 µl of diluted DMSO to each well of the negative control (resting neutrophils) wells.
      5. Incubate for 3 h in a 37 °C incubator.
      6. Add 100 µl 4% PFA to each well without removing the medium (i.e., at final concentration of 1%) to fix the cells at 4 °C for overnight.
    2. Staining with anti-DNA/Histone H1 antibody and DAPI on the next day (Figures 2A and 2B).

      Figure 2. NET demonstration by immunostaining. 2 x 105 human neutrophils (A) or murine neutrophils (B) were activated by 100 ng/ml PMA for 3 h in wells of glass chamber slide. Resting neutrophils with DMSO served as negative control. Slides were fixed and labelled with anti-DNA/Histone H1 antibody followed by AF555-conjugated anti-mouse IgG (red) and nuclei were counterstained with DAPI (blue) (steps B1 and B2). C. Immunostaining of human neutrophil marker CD15 (green) and NET marker DNA/Histone H1 (red) on paraffin-embedded skin section from human vasculitis patients. D. Immunostaining of the murine neutrophil marker Ly6G (green) and NET marker DNA/Histone H1 (red) on paraffin-embedded skin section from mice with induced immune complex-mediated vasculitis (step B3). Scale bars = 100 µm.

      1. Remove medium, add 100 µl PBS to each well and then remove.
      2. Add 100 µl blocking buffer (see Recipe 3) to each well. The slides were left still on bench at room temperature for 45 min.
      3. Dilute DNA/Histone H1 antibody in antibody diluent (see Recipe 4) to a final concentration of 4 µg/ml.
      4. Add 100 µl diluted antibody to each well, incubate on bench at room temperature for 1 h.
      5. Remove antibody, wash slides twice with 100 µl PBS in each well, on an orbital shaker at 100 RPM for 5 min for each time.
      6. Dilute secondary anti-mouse-AF555 1:200 in antibody diluent.
      7. Add 100 µl diluted secondary antibody to each well, incubate at room temperature in the dark for 45 min.
      8. Remove antibody, wash 2 times with 100 µl PBS on an orbital shaker.
      9. Dilute DAPI 5 mg/ml stock 1:5,000 in PBS, add 100 µl to each well, incubate at room temperature in the dark for 3 min.
      10. Remove DAPI staining solution, and remove the frame of chamber slides. Wash slides 2 times by dipping in a container with PBS.
      11. Mount cover slips with fluorescence mounting medium.
      12. Fluorescence microscopy with red channel (AF555) and blue channel (DAPI) (e.g., Zeiss Axiophot microscope with an AxioCam digital color camera and AxioVision software) (see Note 3).
    3. Optional: staining with additional neutrophil marker (Figures 2C and 2D)
      Additional neutrophil markers can be co-stained with NET, in order to provide an additional confirmation. The following primary antibodies have been tested, and one or more combinations can be considered depending on the number of color filters available on the fluorescence microscope: CD15 (human, Novus, 1:50), Ly6G (mouse, Abcam, 1:100), MPO (human and mouse, R&D Systems, 1:50), or NE (human and mouse, Abcam, 1:200). Proper secondary antibodies with different fluorophores other than NET staining are necessary. This staining can be performed before the NET staining. Sequential staining is recommended based on the staining specificity and intensity.
      1. After blocking (step B2b), add 100 µl diluted primary antibody to each well, incubate at 4 °C overnight.
      2. Remove primary antibody, wash 2 times with 100 µl PBS.
      3. Add 100 µl diluted secondary antibody to each well, incubate at room temperature in the dark for 1 h.
      4. Remove secondary antibody, wash 2 times with 100 µl PBS.
      5. Continue with NET staining (step B2c).

  3. NET quantification with Sytox orange (Figure 3)
    Note: If the aim is to study the interaction of specific adherent cells with neutrophils and NET formation, seed the cells of interest one day prior to neutrophil isolation into a 96-well, black, flat bottom plate in 100 µl medium per well, and culture overnight. The seeding cell number depends on the ratio of the target cells to neutrophils. Make 5 repeated wells for each condition. Remove culture medium before adding neutrophils.

    Figure 3. 1 x 105 human neutrophils (hNeu) were cultured alone or with adipose tissue derived mesenchymal stem cells (MSC) at hNeu:MSC ratios of 1,000:1, 100:1 and 10:1 and subsequently activated with 100 ng/ml PMA for 3 h. Cocultures were labelled with 0.25 µM Sytox orange for 5 min at room temperature and fluorescence intensities were measured by a microplate reader. Culture medium containing Sytox orange served as background fluorescent control. Fluorescence intensities were normalized on the intensity of non-activated controls. Results are expressed as mean ± SEM of quintuplicate measurements. This experiment was independently repeated three times with similar results. **P < 0.01; ***P < 0.001; RFI, relative fluorescence intensity; AU, arbitrary unit.

    1. Add freshly isolated neutrophils into a 96-well, black, flat bottom plate at a density of 1 x 105 neutrophils in 100 µl R1 medium (see Recipe 2) per well. A background fluorescence control with only R1 medium, a positive control with PMA activation, and a negative DMSO-only control of resting neutrophil are necessary.
      Note: Make 5 repeated wells for each condition. Avoid the wells in the first and last rows/columns of the plate. (We had experience that the peripheral wells of the plate may give inaccurate fluorescence reading, especially the wells on four corners. It could be due to the technical specification of the microplate reader or due to the quality of the black bottom 96-well plate.)
    2. Add the treatment of interest (see Note 1 in Notes section).
    3. To the positive control wells, and conditions that NET formation are desired, add PMA at a final concentration at 100 ng/ml to activate neutrophils. (i.e., dilute 1 mg/ml PMA stock 1:100 in R1 medium, then add 1 µl of diluted PMA to each well).
    4. Dilute DMSO 1:100 in R1 medium, add 1 µl of diluted DMSO to each well of the negative control wells.
    5. Incubate for 3 h in a 37 °C incubator.
    6. Dilute 5 mM Sytox orange stock 1:5,000 in R1 medium. Then add 50 µl of 1 µM working solution to each well with a multichannel pipette (i.e., Sytox orange at a final concentration of 0.25 µM).
    7. Incubate for 5 min at room temperature in the dark.
    8. Centrifuge the plate at 400 x g for 5 min, carefully remove 100 µl supernatant from each well to reduce the background fluorescence.
      Note: Remove the medium slowly and carefully, and avoid touching the bottom of the plate.
    9. Immediately measure the fluorescence intensity with a Mithras LB940 microplate reader, at excitation and emission wavelengths of 540 nm and 580 nm, respectively (see Note 4).

Data analysis

  1. Calculation of fluorescence intensity of Sytox orange assay
    1. Take the average of 5 repeats of R1 medium only condition. This is the background fluorescence of Sytox orange in medium.
    2. Subtract this background fluorescence from the fluorescence intensity readings of all other samples.
    3. Take the average of 5 repeats for each condition.
    4. Normalize the fluorescence intensity of each condition to the negative control (resting neutrophils), which is set as 1 arbitrary unit (AU).
  2. This assay should be performed at least 3 times with independent neutrophil samples. Data can be presented as mean ± SD (or SEM). Unpaired, two-tailed Student’s t-test can be used for significance test.


  1. The duration of treatment needs to be determined for each individual treatment condition. To study the effect of the target cells on NET formation, a proper incubation time should be considered to allow interaction of studying cells with neutrophils before adding the activator such as PMA. For example, to study the effect of mesenchymal stem cells (MSC) or human dermal fibroblasts (HDF) on NET formation, neutrophils are co-cultured with MSC or HDF for 2 h in R1 medium in a 37 °C incubator (Jiang et al., 2016).
  2. PMA is used to artificially trigger NET formation, for example, as the positive control. In conditions where the suppression of NET formation of the potential treatments is under investigation, the neutrophils are artificially activated with PMA too. However, if the potential treatments are expected to induce NET formation (e.g., bacterial), the addition of PMA is not necessary.
  3. The immunostaining pattern of NET looks different in human and mouse neutrophils. The NET fibers from human neutrophils are much longer and display more prominent network structure. Mouse bone marrow neutrophils are less capable of NET formation, but could be augmented by adding calcium to activate citrullination of histones which are important for NETosis. Regarding few nuclei in Figure 2B, based on our experience, compared to human neutrophils, smaller fraction of mouse neutrophils undergo NETosis. Oxidative burst and other activation processes lead to cell death and not adhering to the dishes.
  4. The microplate reader CLARIOstar with a monochromator from BMG Labtech (Ortenberg, Germany) has been tested for the Sytox orange assay, and it works nicely with excitation at 547 nm, and emission at 570 nm.
  5. The viability and purity of human neutrophils isolated from buffy coat depend on the freshness of the sample. Within 12 h of collection, the expected viability of neutrophils is higher than 80%. The expected purity from both gradient centrifugation and MACSxpress methods is higher than 80%. No difference is noticed in the functional response of neutrophils isolated by the two methods.
  6. In general, it is recommended to use different quantification methods to evaluate NETs, and to combine it with immunofluorescence microscopy to visualize NETs. The Sytox orange assay is useful as an easy NET quantification method for in vitro experiments. For in vivo conditions, a demonstration of neutrophil marker together with antibodies against NET components that can detect decondensed chromatin is recommended.


  1. FACS buffer (can be kept at 4 °C for 2 weeks)
    PBS containing 0.5% FBS, 0.02% NaN3, pH 7.4
  2. R1 medium (prepared media can be kept at 4 °C for 2 weeks)
    RPMI1640 medium
    1% FBS
    1x GlutaMAX
    1x MEM NEAA
    1x penicillin/streptomycin
  3. Blocking buffer (prepare fresh)
    PBS containing 5% BSA together with 5% goat serum
  4. Antibody diluent (prepare fresh)
    PBS containing 1% BSA


This work was supported in part by research grants from the Baden-Württemberg Stiftung (P-BWS-ASII/15), the European Commission (CASCADE HEALTH-FP7-223236) and the German Research Foundation (SFB1149) to K.S.-K., the Baustein Program from the Medical Faculty, University of Ulm (LSBN.0100) to D.J., and from the Excellence Cluster Cardio-pulmonary System (ECCPS) to M.S. Part of figures are adapted and modified from the recent study of Jiang et al., 2016.


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  12. Yu, Y. and Su, K. (2013). Neutrophil extracellular traps and systemic lupus erythematosus. J Clin Cell Immunol 4 (2): 139.


近十年来,嗜中性粒细胞胞外捕获物(NETs)已被鉴定并确认为一种新的抗微生物中性粒细胞武器。 在该方案中,我们描述了通过使用抗DNA /组蛋白H1抗体的细胞外染色质纤维的免疫荧光染色和通过使用非细胞可渗透的DNA特异性染料Sytox orange来定量NETs的简便方法来证明NET形成。
【背景】嗜中性粒细胞构成循环白细胞中最大的进化保守部分。他们通过各种机制建立了针对病原体的第一道防线,包括形成嗜中性粒细胞胞外捕获物(NETs)。在此过程中,活化的嗜中性粒细胞从细胞核中排出染色质纤维。入侵的病原体然后被捕获在染色质网络内并被高度浓缩的NET缠结的抗微生物蛋白如髓过氧化物酶(MPO)和弹性蛋白酶(Brinkmann等人,2004)杀死。然而,NETs是一把双刃剑;来自过度活化的嗜中性粒细胞的无限制的NET形成也可能导致严重的组织损伤,例如通过NET的组蛋白组分的细胞毒性作用(Saffarzadeh等人,2012)。中性粒细胞过度活化并具有增强的形成NETs的能力的病理状况的一个例子是系统性红斑狼疮。狼疮患者血清中针对双链DNA及其它组分的抗体水平升高(Knight and Kaplan,2012; Yu and Su,2013)。来自狼疮患者的受影响的皮肤和肾脏被净化的嗜中性粒细胞渗透,其导致内皮细胞损伤,这是狼疮和其他嗜中性粒细胞过度活化综合征发病的关键步骤(Villanueva等人,2011)。
已经使用不同的方法用于NET检测和定量,包括免疫细胞化学(Brinkmann等人,2010),荧光染料,流式细胞术和ELISA。免疫细胞化学与DAPI和DNA /组蛋白是NET鉴定和定量的最佳方法,因为细胞核解离表明NET形成。 Picogreen染料是量化NET-DNA浓度的敏感方法(Saffarzadeh等人,2012; Tanaka等人,2015),而Sytox橙是一种快速而简单的方法NET定量方法。通过测量标记的DNA /组蛋白抗体的信号或基于髓过氧化物酶 - DNA的ELISA的流式细胞术分析是用于在诸如患者的血清或腹膜液的病理样品中检测和定量NET的有用方法(Caudrillier等人, ,2012)。
在本协议中,我们提供了使用抗DNA /组蛋白H1抗体的染色质纤维的荧光免疫染色来显示NET形成的细节,该抗体与DAPI或Hoechst(Saffarzadeh)相比,在NET中具有非常高的脱胶染色质亲和力等人,2012)。此外,我们描述了使用Sytox Orange(非细胞可渗透的DNA特异性染色染色细胞外DNA含量)进行NET定量的快速方法(Williams等人,1999; Yost等人, ,2009),通过酶标仪测量的荧光强度。该方案已经在我们最近的研究中成功应用,从而我们显示抗体或补体诱导的吞噬作用引发快速的NET形成(Saffarzadeh等人,2014),更重要的是间充质干细胞抑制来自过度活化的嗜中性粒细胞的NET形成(Jiang等人,2016)。

关键字:NETs(中性粒细胞胞外诱捕网), 中性粒细胞, Sytox橙色, DNA/组蛋白H1


  1. 移液器提示(Greiner Bio One International,Ultratip)
  2. 管50毫升(SARSTEDT,目录号:62.547.254)
  3. 注射器10 ml(B.Barun medical,目录号:4606108V-02)
  4. 针26 G x½“(B. Braun医疗,目录号:4665457-02)
  5. 预分离过滤器(30μm)(Miltenyi Biotec,目录号:130-041-407)
  6. Millicell EZ Slide 8孔玻璃(EMD Millipore,目录号:PEZGS0816)
  7. 96孔,黑色,平底板,无菌,带盖(Corning,目录号:3916)
  8. C57BL / 6J小鼠,优选年龄在8-12周龄,男性和女性均适合本方案(“JACKSON LABORATORY”,目录号:000664)
  9. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  10. 0.5M EDTA,无菌(Thermo Fisher Scientific,Invitrogen TM,目录号:15575020)
  11. Histopaque-1077(Sigma-Aldrich,目录号:10771)
  12. Histopaque-1119(Sigma-Aldrich,目录号:11191)
  13. 可选择性:FITC缀合的小鼠抗人CD15抗体(克隆VIMC6)(Miltenyi Biotec,目录号:130-081-101)
  14. 可选:FITC偶联小鼠IgM同种型对照(Miltenyi Biotec,目录号:130-093-178)
  15. 可选:MACSxpress嗜中性粒细胞分离试剂盒,人(Miltenyi Biotec,目录号:130-104-434)
  16. 可选:MACSxpress红细胞消耗试剂盒,人(Miltenyi Biotec,目录号:130-098-196)
  17. 小鼠嗜中性粒细胞分离试剂盒(Miltenyi Biotec,目录号:130-097-658)
  18. 可选:碘化丙啶(PI)染色溶液(BD,BD Biosciences,目录号:556463)
  19. 佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P8139)
  20. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  21. 16%(w / v)多聚甲醛(PFA),无甲醇(Thermo Fisher Scientific,目录号:28908)
  22. 纯化的小鼠抗DNA /组蛋白H1抗体(EMD Millipore,目录号:MAB3864)
  23. 4',6-二脒基-2-苯基吲哚,二盐酸盐(DAPI)(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  24. Alexa Fluor 555缀合的山羊抗小鼠IgG二抗(Thermo Fisher Scientific,Invitrogen TM,目录号:A-21422)
  25. 可选:APC结合的大鼠抗小鼠Ly6G(Gr-1)抗体(克隆RB6-8C5)(Thermo Fisher Scientific,eBioscience TM,目录号:17-5931-82)
  26. 可选择性:APC结合的大鼠IgG2b同种型对照(克隆eB149 / 10H5)(Thermo Fisher Scientific,eBioscience TM,目录号:17-4031-82)
  27. 可选:纯化的大鼠抗小鼠Ly6G抗体(克隆RB6-8C5)(Abcam,目录号:ab25377)
  28. 可选:纯化的兔抗人CD15抗体(克隆SP159)(Novus Biologicals,目录号:NBP2-21754)
  29. 纯化的山羊抗人/小鼠髓过氧化物酶/ MPO抗体(R&amp; D Systems,目录号:AF3667)
  30. 纯化的小鼠IgG2a同种型对照(克隆C1.18.4)(BD,BD Biosciences,目录号:550339)
  31. 纯化兔抗人/小鼠嗜中性粒细胞弹性蛋白酶抗体(Abcam,目录号:ab68672)
  32. Sytox orange核酸染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S11368)
  33. 胎牛血清(FBS)(Biochrom,目录号:S 0615)
  34. 叠氮化钠(NaN 3 3)(Sigma-Aldrich,目录号:S2002)
  35. RPMI 1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:21875034)
  36. GlutaMAX(Thermo Fisher Scientific,目录号:35050038)
  37. MEM非必需氨基酸(NEAA)(Thermo Fisher Scientific,Gibco TM,目录号:11140035)
  38. 青霉素/链霉素(Biochrom,目录号:A 2213)
  39. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  40. 山羊血清(Sigma-Aldrich,目录号:G9023)
  41. 荧光安装介质(Agilent Technologies,DAKO,目录号:S302380-2)
  42. FACS缓冲液(可在4℃保存2周)(参见配方1)
  43. R1培养基(制备的培养基可以在4℃保存2周)(参见方法2)
  44. 阻塞缓冲液(准备新鲜)(参见配方3)
  45. 抗体稀释液(准备新鲜)(参见配方4)


  1. 移液器
  2. 离心机
  3. 可选:MACSxpress分离器(Miltenyi Biotec,目录号:130-098-308)
  4. 可选:MACSmix TM管旋转器(Miltenyi Biotec,目录号:130-090-753)
  5. 加湿细胞培养箱设定为37℃和5%CO 2
  6. 轨道摇床,如Heidolph Unimax 1010(Heidolph Instruments,型号:Unimax 1010,目录号:543-12310-00)
  7. 荧光显微镜,如蔡司Axiophot显微镜与AxioCam数字彩色相机和AxioVision软件v4.7(卡尔蔡司,型号:Axiophot)
  8. AxioCam数码彩色相机
  9. 可测量分别为547nm和570nm的Sytox橙的吸光度和发射率的微孔板读数器,如Mithras LB940(BERTHOLD TECHNOLOGIES,型号:Mithras LB 940)
  10. 可选:流式细胞仪(检查分离的嗜中性粒细胞的纯度)FACS Canto II(BD,BD Biosciences,型号:BD FACSCANTO II)与FACSDiva软件


  1. AxioVision软件
  2. 可选:FACSDiva软件


  1. 嗜中性粒细胞分离
    1. 使用梯度离心法从新鲜的外周血中分离人嗜中性粒细胞(图1A和1B)
      1. 用含有2mM EDTA的PBS稀释外周血1:2。
      2. 在每个50ml管中,仔细并顺序地放置10ml Histopaque-1119,10ml Histopaque-1077和25ml稀释的血液。
      3. 在室温下离心30分钟,在700℃离心(注意:不断裂。)。
      4. 将Histopaque-1077和Histopaque-1119的界面上的层转移到新的50ml管(通常的体积范围为2-3ml)中,用PBS加至50ml,并以300xg离心>室温10分钟。
      5. 用30 ml PBS再次洗涤。

        图1.中性粒细胞分离。 A.通过梯度离心从健康供体的新鲜外周血中分离人嗜中性粒细胞(步骤A1)。收集H1077和H1119界面处的嗜中性粒细胞层。 PBMC,外周血单核细胞; PMN,多形核白细胞;红细胞,红细胞。 B.用PI染料分析细胞活力,并使用PI阴性门内的人CD15抗体分析活嗜中性粒细胞的纯度。 C.用MACSxpress法从血沉棕黄层中分离人嗜中性粒细胞(步骤A2)。用PI染料分析细胞活力,并用PI阴性门内的人CD15抗体分析活细胞嗜中性粒细胞的纯度。 D.使用负耗尽方法通过磁选分离小鼠骨髓嗜中性粒细胞(步骤A3)。用PI染料分析细胞活力,用PI阴性门内的小鼠Ly6G(Gr-1)抗体分析活细胞嗜中性粒细胞的纯度。灰色直方图:PI染料或CD15抗体或Gr-1抗体;开放直方图:未染色的对照或同种型抗体
    2. 替代方法:用MACSxpress嗜中性粒细胞分离试剂盒分离人嗜中性粒细胞(图1C)
      1. 混合5ml的血沉棕黄层(注意:收集后不到12小时。)与1ml重组缓冲液A(试剂盒组分),1ml缓冲液B和100μl重构的MACSxpress红细胞消耗试剂混合。
      2. 使用MACSmix管旋转器以12rpm在室温下孵育样品5分钟。
      3. 将开管放在MACSxpress分离器的磁场中10分钟,然后小心地收集含有嗜中性粒细胞的上清液到新管中。
    3. 鼠骨髓嗜中性粒细胞的分离(图1D)
      1. 解剖C57BL / 6J小鼠的股骨,并通过使用10ml注射器和26G针无菌冲洗出含有2mM EDTA的10ml PBS的骨髓。
      2. 用30微米的过滤器过滤悬浮液。
      3. 按照制造商的说明,用小鼠嗜中性粒细胞分离试剂盒(Miltenyi),使用负耗竭方法通过免疫磁性分离(MACS)分离嗜中性粒细胞。
        注意:8-12周龄的一名C57BL / 6J小鼠的两只股骨骨髓中性粒细胞的平均产量预计为10-15万个细胞。
    4. 可选:检查分离的嗜中性粒细胞的纯度和活力
      1. 在含有5μl抗人CD15-FITC抗体(或小鼠嗜中性粒细胞的抗小鼠Gr1-APC抗体)的100μlFACS缓冲液(参见方案1)中重悬2×10 5个分离的人嗜中性粒细胞。另外在含有5μl小鼠IgM-FITC同种型对照(或小鼠嗜中性粒细胞的大鼠IgG2b-APC同种型对照)的100μlFACS缓冲液中的另外2×10 5个细胞。在室温下在黑暗中孵育30分钟。
      2. 加入100μlFACS缓冲液,轻轻混合,离心,在室温下用3,000 RPM(870 x g,87 mm转子)的台式离心机收集细胞沉淀,除去上清液。
      3. 将沉淀重悬于300μlFACS缓冲液中,加入3μl碘化丙啶(PI)染色溶液,快速混匀
      4. 继续流式细胞仪分析CD15 + PI - 细胞(人)或Gr-1 PI - 细胞(小鼠)。

  2. NET免疫染色显示
    注意:如果目的是研究特定贴壁细胞与嗜中性粒细胞和NET形成的相互作用,则将嗜中性粒细胞分离前一天将感兴趣的细胞种植到8孔玻璃室载玻片(Millicell EZ Slide 8-well glass)在每孔300μl培养基中培养过夜。接种细胞数目取决于靶细胞与嗜中性粒细胞的比例。为每个条件重复井。在添加嗜中性粒细胞前取出培养基
    1. 诱导NET形成
      1. 将新鲜分离的嗜中性粒细胞加入到8孔玻璃室载玻片中,密度为2×10 5个嗜中性粒细胞,每孔在300μlR1培养基(参见方案2)中。为每个条件重复井。
        注意:将中性粒细胞重悬于R1培养基中6.67×10 5细胞/ ml,然后加入每孔300μl。
      2. 添加感兴趣的治疗方法(例如,潜在的化学活化剂/ NET抑制剂,细菌,相互作用细胞,等等)。
      3. 将PMA溶解在DMSO中以制备1mg / ml储备溶液。对于阳性对照孔和需要形成NET的条件,加入终浓度为100ng / ml的PMA以激活嗜中性粒细胞(即,在R1培养基中稀释PMA原液1:100,然后加入3μl稀释的PMA到每个孔)。 (见注2)
      4. 在R1培养基中稀释DMSO 1:100,向阴性对照(静息嗜中性粒细胞)孔的每个孔中加入3μl稀释的DMSO。
      5. 在37℃培养箱中孵育3小时。
      6. 向每个孔中加入100μl4%PFA,而不用去除培养基(即终浓度为1%)即可将细胞固定在4℃过夜。
    2. 第二天用抗DNA /组蛋白H1抗体和DAPI染色(图2A和2B)

      图2.通过免疫染色的NET证明。 2×10 人嗜中性粒细胞(A)或鼠嗜中性粒细胞(B)在孔中被100ng / ml PMA激活3小时的玻璃室幻灯片。休息的嗜中性粒细胞与DMSO作为阴性对照。固定滑动片并用抗DNA /组蛋白H1抗体标记,然后用AF555缀合的抗小鼠IgG(红色)标记,并用DAPI(蓝色)复染细胞核(步骤B1和B2)。 C.人类嗜中性粒细胞标志物CD15(绿色)和NET标记DNA /组蛋白H1(红色)在人类血管炎患者石蜡包埋的皮肤切片上的免疫染色。 D.在具有诱导的免疫复合物介导的血管炎的小鼠的石蜡包埋的皮肤切片上免疫染色小鼠嗜中性粒细胞标记Ly6G(绿色)和NET标记DNA /组蛋白H1(红色)(步骤B3)。比例尺=100μm。

      1. 取出培养基,每孔加入100μlPBS,然后取出
      2. 向每个孔中加入100μl封闭缓冲液(见配方3)。将载玻片在室温下静置45分钟。
      3. 在抗体稀释液中稀释DNA /组蛋白H1抗体(见方案4),终浓度为4μg/ ml。
      4. 向每个孔中加入100μl稀释的抗体,在室温下放置1 h
      5. 去除抗体,每个孔用100μlPBS洗涤载玻片两次,每次在轨道摇床上以100RPM冲洗5分钟。
      6. 稀释次级抗小鼠AF555 1:200抗体稀释液
      7. 向每个孔中加入100μl稀释的二抗,在室温下在黑暗中孵育45分钟。
      8. 去除抗体,用100μlPBS洗涤2次,在轨道摇床上
      9. 稀释DAPI 5mg / ml储备液在PBS中1:5,000,向每个孔中加入100μl,在室温下在黑暗中孵育3分钟。
      10. 取出DAPI染色溶液,取出腔室载玻片的框架。用PBS浸泡在容器中2次清洗。
      11. 用荧光安装介质安装盖子。
      12. 具有红色通道的荧光显微镜(AF555)和蓝色通道(DAPI)(例如,具有AxioCam数字彩色照相机和AxioVision软件的Zeiss Axiophot显微镜)(参见注3)。
    3. 可选:用额外的嗜中性粒细胞标记染色(图2C和2D)
      额外的嗜中性粒细胞标记可以与NET共染色,以提供额外的构象。已经测试了以下一抗体,并且可以根据荧光显微镜上可得到的滤色剂数目考虑一种或多种组合:CD15(人,Novus,1:50),Ly6G(小鼠,Abcam,1:100) ,MPO(人和鼠,R&amp; D Systems,1:50)或NE(人和小鼠,Abcam,1:200)。除了NET染色之外,具有不同荧光团的适当的二次抗体是必需的。该染色可以在NET染色之前进行。建议基于染色特异性和强度进行连续染色。
      1. 封闭后(步骤B2b),向每个孔中加入100μl稀释的一抗,在4℃下孵育过夜
      2. 取一级抗体,用100μlPBS洗2次
      3. 向每个孔中加入100μl稀释的二抗,在室温下在黑暗中孵育1小时
      4. 取出二抗,用100μlPBS洗2次
      5. 继续进行NET染色(步骤B2c)。

  3. NET定量与Sytox橙(图3)

    图3. 1 x 10 5 人类嗜中性粒细胞(hNeu)单独或与脂肪组织来源的间充质干细胞(MSC)在hNeu:MSC比例为1,000:1,100:1和10:1,随后用100ng / ml PMA活化3小时。将共培养物在室温下用0.25μMSytox橙标记5分钟,荧光强度通过酶标仪测量。含有Sytox橙的培养基作为背景荧光对照。荧光强度根据非活化对照的强度进行归一化。结果表示为五次测量的平均值±SEM。该实验独立重复三次,结果相似。 ** 0.01; *** 0.001; RFI,相对荧光强度; AU,任意单位。

    1. 将新鲜分离的嗜中性粒细胞以100μlR1培养基(参见方案2)每孔以1×10 5个嗜中性粒细胞的密度添加到96孔黑色平底板中。只有R1培养基,具有PMA活化的阳性对照和仅有嗜中性粒细胞阴性的DMSO的对照的背景荧光对照是必要的。
      注意:为每个条件重复5个井。避免在板的第一行和最后一行/列中的井。 (我们的经验是,板的外围孔可能会给出不准确的荧光读数,特别是四角的孔,这可能是由于酶标仪的技术规格或黑底96孔板的质量。 )
    2. 添加感兴趣的治疗(见注释部分注1)
    3. 对阳性对照孔和需要形成NET的条件,加入终浓度为100ng / ml的PMA以活化嗜中性粒细胞。 (即,,在R1培养基中稀释1mg / ml PMA原液1:100,然后向每个孔中加入1μl稀释的PMA)。
    4. 在R1培养基中稀释DMSO 1:100,向阴性对照孔的每个孔中加入1μl稀释的DMSO。
    5. 在37℃培养箱中孵育3小时。
    6. 在R1培养基中稀释5mM Sytox橙色储备液1:5,000。然后使用多通道移液管(即终浓度为0.25μM的Sytox橙),向每个孔中加入50μl的1μM工作溶液。
    7. 在黑暗中室温孵育5分钟。
    8. 将板以400×g离心5分钟,小心地从每个孔中除去100μl上清液以降低背景荧光。
    9. 立即用Mithras LB940微孔板读数器测量荧光强度,激发和发射波长分别为540 nm和580 nm(见注4)。


  1. Sytox橙色测定荧光强度的计算
    1. 取平均5个R1中等重复条件。这是Sytox橙在培养基中的背景荧光
    2. 从所有其他样品的荧光强度读数中减去此背景荧光
    3. 每个条件平均5次重复。
    4. 将每个条件的荧光强度归一化为阴性对照(静息嗜中性粒细胞),其被设置为1个任意单位(AU)。
  2. 该测定应使用独立嗜中性粒细胞样品进行至少3次。数据可以以平均值±SD(或SEM)表示。无配对的双尾学生的 t测试可以用于显着性测试。


  1. 需要为每个治疗条件确定治疗的持续时间。为了研究靶细胞对NET形成的影响,应考虑适当的孵育时间,以允许在添加活化剂如PMA之前研究细胞与嗜中性粒细胞的相互作用。例如,为了研究间充质干细胞(MSC)或人真皮成纤维细胞(HDF)对NET形成的影响,在37℃的培养箱中将嗜中性粒细胞与MSC或HDF共培养2 h, > et al。,2016)。
  2. PMA用于人为触发NET形成,例如,作为阳性对照。在抑制潜在治疗的NET形成的条件下,中性粒细胞也被PMA人工激活。然而,如果潜在的治疗预期会诱导NET形成(例如,细菌),则不需要添加PMA。
  3. NET的免疫染色模式在人和小鼠嗜中性粒细胞中看起来不同。来自人嗜中性粒细胞的NET纤维长得多,并且显示更加突出的网络结构。小鼠骨髓嗜中性粒细胞的NET形成能力较弱,但可通过添加钙来激活组氨酸的瓜氨酸化来增强,这对于NETosis很重要。关于图2B中的几个细胞核,根据我们的经验,与人嗜中性粒细胞相比,小部分小鼠嗜中性粒细胞经历NETosis。氧化爆发和其他激活过程导致细胞死亡,而不粘附于盘子。
  4. 已经对来自BMG Labtech(Ortenberg,Germany)的单色仪的微孔板阅读器CLARIOstar进行了Sytox橙色测定,并且在547nm的激发和570nm的发射下很好地工作。
  5. 从血沉棕黄层分离的人嗜中性粒细胞的活力和纯度取决于样品的新鲜度。在收集的12小时内,嗜中性粒细胞的预期存活率高于80%。梯度离心和MACSxpress方法的预期纯度都高于80%。在两种方法分离的嗜中性粒细胞的功能反应中没有观察到差异
  6. 一般来说,建议使用不同的量化方法来评估NETs,并将其与免疫荧光显微镜结合以显现NETs。 Sytox orange测定法可用作体外实验的简单NET定量方法。对于体内条件,推荐使用嗜中性粒细胞标志物以及可以检测脱色染色质的NET组分抗体的证明。


  1. FACS缓冲液(可在4℃保存2周)
    含有0.5%FBS,0.02%NaN 3,pH 7.4的PBS
  2. R1培养基(制备的培养基可以在4℃保存2周) RPMI1640培养基
    1x GlutaMAX
    1x MEM NEAA
  3. 阻塞缓冲区(准备新鲜)
  4. 抗体稀释剂(准备新鲜)


这项工作部分得到巴登 - 符腾堡基金会(P-BWS-ASII / 15),欧洲委员会(CASCADE HEALTH-FP7-223236)和德国研究基金会(SFB1149)向KS-K提供的研究资助。来自乌尔姆大学医学院(LSBN.0100)至DJ的Baustein计划,以及卓越集群心肺 - 系统(ECCPS)至MS一些数字是从近期江泽民的研究报告(2016年)进行了调整和修改的。


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引用:Jiang, D., Saffarzadeh, M. and Scharffetter-Kochanek, K. (2017). In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation. Bio-protocol 7(13): e2386. DOI: 10.21769/BioProtoc.2386.