Jul 2020



An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation

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The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker.

Keywords: NETs detection (中性粒胞外菌网的探测), NETs quantification (定量中性粒胞外菌网), Imaging flow cytometry (成像流式细胞术), NETosis (NETosis), Histone H4 citrullination (组蛋白H4瓜氨酸化), Nuclear decondensation (核解凝)


Activated neutrophils are rapidly recruited to sites of infection and injury; they contribute to host defense against pathogens and inflammation. About two decades ago, it was observed for the first time that in response to pathogens, a fraction of the neutrophil population can undergo a distinct type of cellular death, different from apoptosis or necrosis. This involves early chromatin decondensation, co-localization of the nuclear and granular compartments and finally, release of this mix into the environment. The extracellular DNA-scaffolded strands anchoring nuclear components (such as histones) or cellular constituents, some with strong anti-pathogen properties like elastase or myeloperoxidase (MPO), are known as “neutrophil extracellular traps” or NETs and have effective antimicrobial activity (Brinkmann et al., 2004). More recent evidence shows that NETs also associate with non-infectious conditions including cancers, systemic lupus erythematosus, sickle cell disease, atherosclerosis, and thrombosis (Jorch and Kubes, 2017). It is yet not understood whether the NETs, or any of their components (cell-free DNA or histones) contribute to the pathology of these disorders as drivers, or whether they are by-products of an unbalanced immune response. NETs might also function as biomarkers in certain diseases and provide information regarding the efficacy of a treatment regimen (Barnado et al., 2016). NETs-associated components such as elastase or MPO are present in the plasma of individuals with autoimmune diseases and also in infections, which complicates the specificity of these molecules as biomarkers. In this light there is a great need for new methodologies for NETs characterization that offers consistent criteria between studies, and are reliable for both mechanistic and clinical applications.

Other NETs detection and quantification methods are being developed in addition to the classical widely used immunofluorescence microscopy technique. These include use of confocal microscopy that provides information on the structure of the NETs (Santos et al., 2018), ELISA-based assay to quantify the citrullinated histone 3 (H3cit) in human plasma (Thalin et al., 2017), high throughput live detection of NETosis- and apoptosis-related nuclear changes using membrane-permeable and -impermeable nuclear dyes in human neutrophils (Gupta et al., 2018), quantification of free chromatin (predominantly associated with elastase) in human whole blood by using microfluidics (Muldur et al., 2018). Several studies monitored NETs components (H3cit, MPO and extracellular DNA) in human and mice by using conventional flow cytometry (Gavillet et al., 2015; Masuda et al., 2017; Lee et al., 2018; Zharkova et al., 2019). While this technique allows high-throughput analysis it also does have noteworthy limitations, regarding the choice of the probes, the preparation of the samples and the way gating impacts final conclusions (Manda-Handzlik et al., 2016; Masuda et al., 2017). On the other hand, while Imaging Flow Cytometry (IFC) does allow high-throughput analysis, it also combines visualization and analysis features from both conventional flow cytometry and fluorescence microscopy (Basiji, 2016). NETs-associated morphological nuclear changes (i.e., chromatin decondensation and DNA trails) and whether and to what degree MPO co-localized with the nuclear compartment were investigated with IFC (Zhao et al., 2015; Ginley et al., 2017; Pelletier et al., 2017). Thus, different markers and different methods are currently employed to detect and quantify NETs in vitro. PAD4-mediated histone citrullination has been long designated as a hallmark of NETs formation, and thus a desirable marker to follow. More recent reports, however, predominantly in the mouse model, suggest that PAD4-mediated citrullination of histone 3 (H3cit) is stimulus-dependent (Neeli and Radic, 2013; Claushuis et al., 2018; Guiducci et al., 2018; Liang et al., 2018) and therefore the utility of this site as a NETs marker might be limited.

Here, we describe in detail a novel IFC protocol that allows specific detection and quantification of citrullination of histone 4 (H4cit3) as a NETs marker in whole neutrophils, prior to the release of the DNA and the cytoplasmic protein strands into the extracellular space. Other analysis parameters that look at nuclear and cellular morphological changes (nuclear decondensation and supercondensation, multi-lobulated nuclei and cell membrane damage) can bring additional information on the behavior of the analyzed neutrophil population. To establish our methodology we determined the responses of healthy human neutrophils treated for 5 different periods of time (between 2 min and 60 min) with NETs agonists: pharmacological inducers, PMA and calcium ionophore (a well-known inducer of histone citrullination, and for our experimental conditions, the positive control), Hemin (an inducer present under hemolytic conditions) and LPS and IL-8 (inducers associated with infectious pathogens). We used in vitro fluorescence microscopy to confirm the formation of DNA-elastase-MPO strands in healthy neutrophils treated with the stimuli used for the IFC tests (Barbu et al., 2020). We further validated this technique in untreated and Hemin-treated neutrophils from healthy donors and patients with sickle cell disease at steady state (Barbu et al., 2019).

The increased sensitivity and objective analysis make this methodology highly suitable for NETs detection in both research and clinical studies, that explore mechanistic answers or possible therapeutic strategies.

This protocol has 4 major parts: one-step neutrophil purification, induction of NETosis, specific staining, IFC acquisition and analysis (as highlighted in Figures 1A and 1C), with the first 3 steps requiring 3 days and up to one week to complete all steps.

Figure 1. Day-by-day description of the major steps for specific detection of NETosis markers by Imaging Flow Cytometry. A. One-step polymorphoponuclear (PMN) cells isolation from whole blood with Polymorphprep medium; with this method neutrophils purity should be consistently above 80%. Total processing time: 90 min. B. An example of correct separation of whole blood by using Polymorphprep gradient medium. C. Detailed description of polymorphonuclear cells treatment and staining procedure. A time recommendation is not included for the neutrophil stimulation step as researchers should choose an appropriate treatment time according to their experimental purpose. Each washing step might need up to 20 min to complete, depending on the number of samples processed. This time should be taken into account while calculating total time required to complete all steps per day. Short final wash followed by samples storage at 4 °C. Total stimulation and staining processing time for Day 1: 4+ h.

Materials and Reagents

  1. See Through, Lavender, 10.0 V, 16 × 100 mm, Plastic Tube with BD Hemograd Closure (BD, catalog number: BD366643)

  2. Polypropylene Conical Tubes, 50 ml (Fisher Scientific, Corning, catalog number: 07-203-510)

  3. Centrifuge tubes, 5 ml, conical bottom, sterile (Benchmark Scientific, catalog number: C1005-T5-ST)

  4. Centrifuge tubes 1.5 ml, mixed neon colors (USA Scientific, catalog number: 1415-1448)

  5. Rapid flow sterile disposable filter units with PES membrane, capacity 500 ml, pore size 0.45 μm (Thermo Scientific Nalgene, catalog number: 09-740-63E)

  6. Aluminum foil

  7. Polymorphprep gradient medium (Cosmo Bio USA, catalog number: AXS-1114683)

    Note: Polymorphprep should be stored at room temperature, in the dark. Long term exposure to light affects its efficacy.

  8. RPMI-1640 without L-glutamine (Lonza, catalog number: 12-167F)

  9. L-glutamine 200 mM (Thermo Fisher, catalog number: 25030-081)

    Note: Aliquot in one-time use fractions and store at -80 °C. Thaw in water bath at 37 °C to dissolve the white sediment, then add to the RPMI to make the complete neutrophil medium.

  10. Stock 32% Paraformaldehyde (PFA) Aqueous Solution, EM Grade (Science Services, catalog number: E15714). Once open use within days

  11. Ultrapure 0.5 M EDTA pH 8.0 (Thermo Fisher, Invitrogen, catalog number: 15575020)

  12. DPBS 1×, no calcium chloride, no magnesium chloride (Gibco, catalog number: 141190-136)

  13. Cell Culture Grade Water (Corning, catalog number: 25-055-CV)

  14. Bovine Serum Albumin, heat shock fraction, protease free, pH 7.0, ≥98% (Millipore Sigma, catalog number A3294)

  15. Potassium Chloride Granular (Mallinckrodt, catalog number: 6858)

  16. Dimethyl Sulfoxide (DMSO), ≥99.5% (GC), plant cell culture tested (Sigma-Aldrich, catalog number: D4540)

  17. Hemin from bovine, ≥90% (Millipore Sigma, catalog number: 9039)

    Note: Prepare a 40 mM stock in DMSO, aliquot in single use fractions and store at at -20 °C. Mix well when preparing the working solutions. Add a DMSO-only control with the tested samples.

  18. PMA, for use in molecular biology applications, ≥99%, HPLC (Millipore Sigma, catalog number: P1585)

    Note: Prepare a stock solution in DMSO and freeze single use aliquots at -20 °C.

  19. LPS-EB, Ultrapure, E. coli 0111:B4 (InvivoGen, catalog number: tlrl-3pelps)

    Note: Prepare a stock solution in ddH2O, aliquot and freeze at -20 °C. Limit the thaw-freeze cycles to three.

  20. Recombinant Human IL-8 (CXCL7, 77aa) (Peprotech, catalog number: 200-08)

  21. Calcium Ionophore A23187 ≥98% (TLC), powder (Millipore Sigma, catalog number: C7522)

  22. Anti-Human CD66b-PE, clone G10F5 (Biolegend, catalog number: 305106)

  23. Rabbit polyclonal anti-histone H4, citrulline 3 (H4cit3, Millipore Sigma, catalog number: 07-596) (see Notes)

  24. Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 680 (Thermo Fisher, catalog number: 35568) (see Notes)

  25. MPO Polyclonal Antibody, AlexaFluor 594 conjugated (Bioss Antibodies, catalog number: bs-4943R-A594)

  26. Hoechst 33342 (BD Pharmingen, catalog number: 561908)

  27. Gelatin from Porcine Skin, powder, Type A, suitable for cell culture (Millipore Sigma, catalog number: G1890)

    Note: Prepare 2% stock and keep it at 4 °C for up to 6 months. Open in sterile hood only. Monitor for signs of bacterial or fungal contamination.

  28. BD Cytofix/Cytoperm kit (BD Biosciences, catalog number: 554722). Use only the Cytofix/Cytoperm solution in this protocol

  29. Wash Buffer (see Recipes)

  30. Blocking buffer (see Recipes)

  31. Paraformaldehyde 8% working solution (see Recipes)

  32. Porcine skin gelatin stock 2% (see Recipes)

  33. 0.6 M KCl (see Recipes)

  34. Neutrophils complete medium (see Recipes)


  1. Water bath at 37 °C

  2. LabGard® ES, Class II, Type A2, Biological Safety Cabinet (NuAire, Plymouth, MN)

  3. Benchtop Centrifuge with swinging bucket rotor (Beckman Coulter, model: Allegra X-14R)

  4. Centrifuge (Thermo Fisher Scientific, model: Sorvall ST 16R)

  5. CO2 Incubator (Thermo Fisher Scientific, model: Heracell VIOS 160i)

  6. Amnis ImageStream Mark II imaging flow cytometer (Luminex Corporation, Austin, TX, USA)


  1. Amnis INSPIRE (Luminex Corporation, Austin, TX, USA) for data acquisition

  2. Amnis IDEAS (Luminex Corporation, Austin, TX, USA) for data analysis, available for download on the company’s website


  1. Neutrophil solation using Polymorphprep medium


    1. Choose a high yield one step neutrophil purification method as this NETs detection protocol requires relatively high number of neutrophils (2 × 106 per sample) with minimal background activation.

    2. The centrifugation step must be performed in a swing bucket rotor as the 500 × g speed has to be reached in the middle of the tube, at the interface between the blood and the Polymorphprep medium.

    3. It is not uncommon that the Polymorphprep separation fails when using blood from female donors. In our experience this separation works consistently with donors of both sexes that have a MHCH (mean corpuscular hemoglobin concentration) value within the normal range.

    4. Keep the purified neutrophils on ice at all times and use them for experiments within 2 h.

    1. Collect whole blood in 10 ml EDTA (purple top) vacutainers and start neutrophil isolation within 1 h of the blood draw.

    2. Equilibrate rotors and adaptors of the centrifuge to room temperature (18 °C to 22 °C). Set “Acceleration” to one; set “Deceleration” to zero. Set centrifugation to 500 × g for 30 min.

    3. Layer, very gently, undiluted whole blood on to the Polymorphprep medium always kept at room temperature, at 1:1 ratio and proceed to centrifugation. Centrifuged tubes will have the following layers from top to bottom: plasma, peripheral blood mononuclear cells – polymorphonuclear cells (neutrophils) – red blood cells (as shown in Figure 1B).

    4. Collect the neutrophil ring and gently re-suspend in 50 ml ice-cold DPBS. Centrifuge at 400 × g for 10 min at 4 °C, with acceleration and deceleration set at maximum. Remove and discard platelets-containing supernatant.

    5. Flick the pellet to resuspend, then add 3 ml ice-cold sterile water and make sure the pellet is fully re-suspended by gently pipetting up-and-down a few times. After 30 s restore osmolarity with 6 ml sterile ice-cold 0.6 M KCl. Fill up with ice-cold DPBS and centrifuge at 330 × g, 7 min at 4 °C.

    6. Discard the supernatant and re-suspend the pellet in complete neutrophil medium equilibrated at room temperature (RPMI supplemented with 2 mM L-glutamine) at 4 × 106 cells/ml.

  2. Induction of NETosis


    1. Adapt the choice of NETs inducers and the length of the treatment according to the experimental requirements.

    2. Distinctive NETs agonists induce optimal responses at different lengths of the treatment period. Five time points (2, 7, 15, 30 and 60 min) and 5 different agonists (calcium ionophore, Hemin, LPS, IL-8 and PMA) have been tested in this protocol.

    3. Allow purified neutrophils to rest before treatment with the NETs inducers to reduce their background activation.

    4. For each experiment prepare fresh 8% PFA dilution in 1× DPBS and keep it cold and in the dark until needed (this will be further diluted to a final concentration of 4% in a further step. Do not store for second use).

    5. All steps below can be performed directly in 1.5 ml Eppendorf tubes if so chosen. However, vigorously mixing the PFA into the cell suspension by pipetting can result in splashing and loss of cells. Using 5 ml tubes provides more space for thorough mixing and to prevent loss of cells.

    1. Add purified neutrophils, 2 × 106 cells (in 500 μl of complete neutrophil medium) in a 5-ml sterile tube and allow the purified neutrophils to equilibrate at room temperature.

    2. Transfer tube to a 37 °C incubator at 5% CO2 and allow them to rest for at least 30 min without closing the cap.

    3. Add 100μl of complete RPMI for the untreated control or the RPMI containing the NETs inducers of choice, mix gently the same number of times in all tubes and return tubes to incubator for the desired periods of time. Do not close the caps of the tubes during this incubation period.

    4. Remove from incubator and stop reaction by adding 600 μl of freshly made 8% PFA in 1× DPBS for a final concentration of 4% PFA, mix at least 5 times by pipetting, and allow to stand at least 30 min at room temperature, cover with aluminum foil.

    5. Add 1ml Wash Buffer (WB) at room temperature and centrifuge at 1,120 × g for 5 min, at 4 °C. Discard the PFA-containing supernatant into the designated chemical waste.
      Note: To ensure that the PFA is removed, repeat this wash step one more time.

    6. Gently re-suspended the pellet with 500 μl of WB and transfer it into 1.5 ml Eppendorf tube for subsequent staining steps.

    7. Repeat the washing of the 5-ml tube with another 500 μl of WB to make sure all cells have been collected for staining.

  3. NETosis staining technique for imaging flow cytometry


    1. Remove the supernatant completely from cells at all steps, while paying attention not to lose cells in the process.

    2. Determine the optimal working dilutions for all the antibodies used in the staining panel by titrating with a range of dilutions above and below of those recommended by the vendors (for example, for a recommended dilution of 1:1,000, up to 5 different dilutions should be tested – 1:250; 1:500, 1:1,000, 1:2,000, 1:3,000). This is required as the lasers on different ImageStream cytometers might have different powers.

    3. Optimal H4cit3 dilution is defined as the concentration that provides the greatest separation of the positive staining against the background (and has highest index score when antibody titration is performed). Because the concentration of the antibody stock provided by the company might vary between lots, the optimal H4cit4 dilution has to be determined for each new lot of antibody.

    4. Acquire the samples within 72 h of the end of the staining protocol. Longer wait times might interfere with the strength of the signal, particularly for Hoechst (which leaks freely from the stained cells with time). Our recommendation is to acquire them immediately.

    5. Store stained cells at 4 °C in the dark and keep them on ice and in the dark during acquisition on the ImageStream cytometer.

    1. In the 1.5 ml Eppendorf tubes re-suspend fixed and thoroughly washed cells in 100 μl of 2% BSA containing CD66b-PE, mix vigorously at least 5 times by pipetting and incubate for 20 min, at room temperature, in the dark.

    2. Washing step: Add 500 μl WB and centrifuge at 1,120 × g for 5 min, at 4 °C. Remove the supernatant and repeat washing two more times.

    3. Re-suspend washed pellet in 300 μl of BD Cytofix/Cytoperm, mix by pipetting up and down at least 5 times, incubate 15 min at room temperature, in the dark.

    4. Washing (step #2).

    5. Re-suspend washed pellet in 500 μl of blocking buffer, mix by pipetting up and down at least 5 times, incubate overnight at 4 °C, in the dark.

    6. Washing (step #2).

    7. Re-suspend in 100 μl of 2% BSA containing anti-histone H4 citrulline 3 (H4cit3) primary antibody at the optimal determined dilution. Pipet up and down at least 10 times and incubate overnight at 4 °C.

      Note: The concentration of this primary antibody is the limiting factor for the final strength of the fluorescence signal (see note b above).

    8. Washing (step #2).

    9. Re-suspend pellet in 100 μl of 2% BSA containing secondary antibody DyLight 680, at the determined optimal dilution and the anti-MPO-AlexaFluor 594 conjugated. Pipet up and down at least 10 times and incubate for 30 min, at room temperature, in the dark.

    10. Washing (step #2).

    11. Re-suspend in 200 μl of 2% BSA containing Hoechst diluted as suggested by the vendor (1:1,000). Mix thoroughly by pipetting and incubate for 15 min, at room temperature, in the dark.

    12. Add 500 μl WB and centrifuge at 1,120 × g for 5 min, at 4 °C. Remove supernatant.

    13. Re-suspend in 50 μl WB for ImageStream (Imaging Flow cytometer) acquisition (see notes d and e above).

  4. Imaging flow cytometry acquisition.

    Within the INSPIRE software, establish the acquisition settings and create a template to be used with all experimental repeats.

    1. Under the “Illumination” tab turn on the 785 laser (side scatter laser – SSC) and all other lasers to be used in the experiment, based on the fluorochromes panel (a chart showing the laser excitation wavelength and its corresponding dyes is available in the company’s website).

    2. Under the “Magnification” section, adjust magnification to 60×.

    3. Under the “Fluidics” section, slide the Speed/Sensitivity bar to the left (to ensure acquisition with low speed and high sensitivity).

    4. Under “File Acquisition”, create a new folder to save the acquired files (with a .rif extension – raw image file).

    5. Load a sample stained with all the colors and expected to yield the brightest signals for all the dyes used (stimulated with PMA as an example – the rational here is to ensure the positive fluorescent signal is within range and will not be saturating the channel).

    6. Adjust the laser voltages based on the maximum of fluorescence without channel saturation of each fluorochrome according to their correspondent laser excitation.

      Note: When setting the channel-specific laser power, the optimal voltage of the laser is the one that does not saturate the detectors (less than 4096 pixels in the generated image) and also promotes a clear distinction between negative and positive population. A ‘Raw Max Pixel’ feature of each of the used channels should be applied. This feature provides the highest value of the pixels contained in the input mask.

    7. Once the laser voltages are set, “Return” the sample and set it aside to re-acquire it after the single color compensation control tubes have been acquired for spillover matrix calculation.

    8. To generate a compensation matrix file, start the compensation wizard on Inspire and acquire at least 1,000 events from each of the compensation control tubes.

      Note: The customers portal on the Amnis ImageStream website provides detailed instructions of how to run and validate compensation matrix using the Wizard in the INSPIRE (acquisition software). A compensation matrix has to be applied to every acquired .rif file when first opened for analysis in the IDEAS software (even when a compensation matrix was also applied during the acquisition of that file with INSPIRE). Spillover compensation can be done during acquisition or post-acquisition of data files. If the stop gates on acquisition of the files are based on fluorescent signals, for example, acquisition based on 5,000 positive cells for CD66b, then it is advisable to perform compensation on INSPIRE (use the compensation wizard) to ensure that the fluorescent signal is corrected for spillover in other fluorescent channels.

    9. Create a dot plot of Area of the Brightfield (BF) versus intensity of Side Scatter (SSC) channels and by clicking around the dots (clusters of populations) and evaluating the correspondent imagery displayed in the gallery, the single cells can be found and boundaries determined. In this protocol, for purified neutrophils, the gate of single cells should be in the range of 50 µm2 to 200 µm2 area. This should exclude most of the debris, small particles (to the bottom and left) and aggregates (slightly upper and right – please refer to Figure 2A).

      Note: Due to the nature of neutrophils, we thought it would be easier to identify singlets by the use of Area versus SSC; however, one can also use Area versus Aspect Ratio (AR) of BF to determine single cells. Aspect Ratio is the ratio of width by the height, events closer to AR of 1 will be where the single events should be located.

    10. From the gate above, create a histogram using the gradient RMS (root mean square) feature of the BF (Channel 1 or 9), to select the cells in focus. Because this feature measures the sharpness quality of an image, changes of pixel values of 40 and above, usually corresponds to the cells in best focus. Selecting the bin and visualizing the correspondent cells within the bin in the image gallery will help determine where to begin the focused gate in this histogram.

    11. Set the “Acquisition” tab to acquire 20,000 single and focused cells (first and second plots created, respectively).

    12. Save this template to be used for subsequent acquisitions with the same settings for laser powers, magnification and number of events acquired.

    13. Type the name of the sample into the “Acquisition” tab.

    14. “Load” the sample, confirm cells are centered and in focus and click “Acquire”.

      Note: Follow one of the two available brightfield channels (channel 1 or channel 9) to confirm the cells are centered (i.e., the cells are flowing in the middle of the channel) and in focus (the images of the flowing cells are clear and crisp).

Data analysis


  1. Representative images for steps 3 to 5 are shown in Figure 2A.

  2. For a full set of experimental results with our tested NETs agonist refer to our previous publication (Barbu et al., 2020).

  1. In the IDEAS analysis software, open the .rif extension file to be analyzed.

  2. Apply the compensation matrix to be used.

    Note: A previously made compensation matrix is stored as a .ctm file. IDEAS software will prompt for selection of a compensation matrix for every .rif file that needs to be analyzed. Navigate to the stored file and load it by using the instructions from the analysis software.

  3. Create a dot plot of area of the BF image (M01 – default mask) versus side scatter (SSC – channel 6).

    Note: Single neutrophils with high SSC and relatively big cellular area will appear as a distinct clustered population. Check a few of the single cells to define the boundaries of the singlets gate and exclude debris and mononuclear cells. The best way to determine and validate the gate boundaries, besides the visible cluster of events in the graph, is to click on some dots in the plot and visualize the correspondent imagery. When looking on Area axis, one should expect single events to the right of the population of debris or smaller cells and to the left of doublets, larger aggregates or even bigger cells, if the cell suspension may contain such cells.

  4. Select the focused cells by using the focus quality feature in brightfield, Gradient RMS (Root Mean Square).

  5. Create a dot plot of signal intensity for granulocyte-specific marker CD66b versus Hoechst and gate double positive events (CD66b+Hoechst+) for further analysis.

  6. In the CD66b+Hoechst+ gate create a histogram for the intensity of the H4cit3 signal and evaluate percent of H4cit3+ cells and/or mean/median intensity fluorescence for this marker (Figure 2B).

    Note: We recommend the use of median intensity fluorescence for this parameter.

  7. On the nuclear channel (Hoechst) the CD66b+Hoechst+ gate create a dot plot of ‘Area’ versus the specific texture feature ‘Bright Detail Intensity_R3” (BDI_R3) to identify neutrophils with decondensed nuclei from those with normal nuclei in the CD66b+Hoechst+ gate (Figure 2C).

    Note: BDI is a feature that calculates intensity of localized bright spots within the masked area in the image (the description of this and other IDEAS features used in this protocol can be found in the downloadable IDEAS ImageStream Analysis Software User’s Manual).

    Figure 2. Gating strategy for identification of neutrophils and their nuclear NETosis markers by using IDEAS software. A. Single cells identified with two-parameter dot-plot of area of Brightfield (BF – X axis) versus Intensity of Side Scatter on Channel 6 (SSC – Y axis) (left panel). Focused singlets cells selected by using Gradient RMS (Root Mean Square) in Brightfield (middle panel). Neutrophils (CD66b+Hoechst+, in strawberry red) identified from singlet focused cells in a two-parameter dot-plot of fluorescence of Hoechst on Channel 7 (X axis) and CD66b on Channel 3 (Y axis) (right panel). B. Representative histograms showing the percentage of H4cit3hi cells in the CD66b+Hoechst+ gate and H4cit3 median fluorescence intensity (MFI). In the right panel the neutrophils were left untreated in RPMI (red) or were treated with 20 uM Hemin for 15 min (green). C. Decondensed nuclei (purple) and normal nuclei (green) identified with a dot-plot of area of nucleus (morphology mask function of Hoechst staining on Channel 7) versus Bright Detail Intensity R3 of the Hoechst staining (examples of nuclei from the two gates are included on the right side of the panel). Neutrophils were left untreated (left) or were treated with 20 μM Hemin for 60 min (right). Percentage of cells with decondensed nuclei is shown. Scale bars = 7 μm.

  8. Use Similarity feature on the nuclear dye channel (Hoechst) and MPO channel to determine the degree to which the two images are linearly correlated within the masked nuclear area in cells with normal and decondensed nuclei (Figure 3A).

    Note: When located in the cytoplasm MPO staining has a dissimilar distribution compared to the Hoechst-stained nuclear mask (i.e., low Similarity score, no co-localization). When the intensity of both dyes at the nuclear location is high, the high Similarity values indicate co-localization.

  9. Use the customized “Level Set” mask on the nuclear dye channel and the ‘LobeCount’ feature to detect reduction in the percentage of cells with multi-lobulated nuclei and separately quantify cells with variable number of lobes (1-lobe to 4-lobes) (Figure 3B).

  10. In the 1-lobe gate create a dot plot of ‘Area of nucleus’ versus ‘Ratio of nuclear by the whole cell areas’ to quantify nuclear supercondensation. Supercondensed nuclei have low values for both parameters; (Figure 3C).

    Note: See the stand-alone Notes chapter for instructions on how to construct this custom feature. For better resolution of the nuclear area, custom masks should be used for the above-mentioned parameters.

  11. In the normal nuclei gate construct a dot plot of ‘Standard deviation’ (X axis) and ‘Modulation’ (Y axis) to assess changes in cell membrane texture. High values for Standard deviation in darkfield (SSC channel) and for Modulation in brightfield indicate neutrophils with damaged membranes (Figure 3D).

    Note: The Standard Deviation Feature provides information on the complexity of an object. Modulation Feature can quantify image quality and thus characterize cells’ texture.

    Figure 3. IDEAS software analysis features to identify and quantify additional nuclear and cellular alterations. A. Similarity analysis feature for DNA (Hoechst – channel 7) and MPO (AF-594 – channel 4) indicates co-localization of the nuclear compartment with type I granules in neutrophils with normal nuclei (green) and decondensed nuclei (purple). Examples of cells with low (< 1) and high (3) similarity scores, indicating low and high DNA-MPO co-localization, respectively, are included (the position of their specific bins on the graph is highlighted in aqua). B. Lobe Count feature identifies changes in the number of nuclear lobes based on nuclear imagery and quantify the number of cells in each category. Representative images of neutrophils with 1 lobe or multilobed nuclei are included. C. Dot-plot of Area of Nucleus (morphology mask of Hoechst staining) on X axis versus ratio of the nucleus by the whole cell areas in the 1 lobe nucleus gate identifies supercondensed nuclei with low nuclear area and consequently low ratio when divided by the whole cell area. Representative images of cells with supercondensed nuclei are presented. D. Neutrophils with damaged membranes (bright green) are distinguished with two-parameter dot-plot of Standard Deviation Side Scatter on Channel 6 (X axis) versus Modulation of Brightfield (Y axis). High scores for both features indicate complex and textured objects. Included, representative imagery of cells within both gates. (BF = Brightfield; H = Hoechst). Scale bars = 7 μm.

  12. Save file once all the analysis parameters have been established. For each .rif acquisition file, two additional files (.daf and .cif) are created in the analysis step. Use the .daf for analysis. Do not remove the .cif one (this is where all analysis data is stored).

  13. Use “Batch Data File” under the “Tools” tab to assign all saved analysis parameters to the remaining .rif file to be analyzed.

  14. In the .daf analysis file use “Reports” to define a statistic report for all parameters of interest in the target subpopulations and then “Generate Statistic Report” to apply it to all analyzed sample files (Figure 4).

    Figure 4. Define and generate a statistic report with all analysis parameters of interest. Use “Add Files” function to apply all defined stats to the .daf analysis files. IDEAS software generates and stores a .txt statistics file that can be opened with Excel for further processing.


  1. In this protocol all steps (neutrophils isolation and stimulation, staining, detection and NETs quantification) can critically impact the data quality.

  2. Neutrophils are notorious for being easily activated during purification. Rest the purified population for 30 min in the incubator prior to the NETs stimulation to reduce false positive data.

  3. Confirm purification of neutrophil population by additional methods (e.g., cytospin).

  4. Stop the NETs challenge step by adding PFA into the reaction mix, rather than spinning the tube and removing the supernatant before adding the PFA to reduce activation by other means.

  5. Confirm the lack of non-specific binding for the secondary antibody (DyLight 680) by running a control tube with cells and secondary DyLight 680 antibody but no H4cit3 primary antibody.

  6. Use custom masks for the data analysis with the IDEAS software, as they represent more accurately the region of interest. Validate the custom masks for total cell area and nucleus, by confirming that the masks are indeed masking the region(s) of interest (i.e., not missing parts of the target region or, on the opposite, masking ‘ghost’ regions outside of what is relevant for the features) (Dominical et al., 2017).

  7. Note that this protocol allows the identification of NETosis markers in whole neutrophils, prior to the release of the DNA and the cytoplasmic proteins strings into the extracellular space. This detail is crucial for deciding the length of the challenge with the NETs inducers for a peak number of cells that respond to stimulus and can be acquired with the ImageStream cytometer. Lengthy stimulation periods might lead to false negative results due to the fact that the cells have already lysed and released their content and thus are no longer “visible” to the flow cytometer. This case can be confirmed by independent assessments: for example, by the size of the pellet observed in the staining tube, and the number of events in the CD66b+Hoechst+ gate actively acquired by the flow cytometer in the stimulated tube being reduced as compared to the untreated control.

  8. To generate a custom feature to evaluate nuclear supercondensation:

    1. In the IDEAS software, open a .daf extension file, select a Hoechst+CD66b+ double positive cells.

    2. On the top menu in IDEAS, select ‘Analysis’, then ‘Masks’. In the open pop-up window select ‘New’, then ‘Function’.

    3. To create a mask for the nuclear staining: select the fluorescent channel for the nuclear imagery for both the image and the channel boxes. In the drop-down menu of the function masks, test which mask function fits best with the nuclear image by looking at the changes of masking in the imaging box (usually “Morphology” works best as nuclear mask, as it covers more accurately the nuclear stained region) (Dominical et al., 2017). Once the new function mask is chosen, Click ‘Ok’.

    4. Create a name for that mask or use the default name suggested by the software.

    5. Click ‘Ok’ again to generate the mask.

    6. To create a new mask press ‘New’.

    7. Repeat steps b through e to mask the whole cell area by using CD66b imagery or BF channel. “Object” or “Erode” function masks are good choices for masking the cell region.

    8. Click ‘Close’ to close the mask window.

    9. To apply new features on these new custom masks created, go back to ‘Analysis’ in the top menu bar and select ‘Features’. Select “New” in the pop-up window.

    10. ‘Feature Type’ box is available for choosing an option. Choose ‘Single’ and in the drop-down menu in the side, choose the ‘Area’ feature.

    11. Below the ‘Feature Type’ box, in the mask box, select the nuclear mask newly created. ‘Set a Default Name’ or create a new name. Click ‘Ok’ when done.

    12. Repeat steps i to k. Next use the ‘whole cell’ mask generated to input in the mask box of the new area feature to create the new ‘Area of the whole cell’ feature.

    13. To calculate the ratio of these new features created, make a combined feature by selecting ‘New’ in that same feature window.

    14. In the ‘Feature Type’ box select ‘Combined’.

    15. In the ‘Features Box’ on the left, select the ‘nuclear area’ feature created.

    16. Highlight the feature and on the right side of the window select the ‘arrow down’ to insert that feature in the box.

    17. Select “/” (forward slash) to calculate the ratio.

    18. Highlight the ‘Area of the whole cell’ new feature created and press the arrow down to insert this feature in the box after the ratio sign. The nuclear area is now divided by the whole cell area.

    19. Name the new feature. Click ‘Ok’ when done.

    20. Click ‘Close’ to close the Features window. This new feature can be now applied in histograms or two-parameters dot plot.


  1. 8% Paraformaldehyde (PFA) working solution

    2.5 ml PFA 32% stock

    Add 10 ml 1× DPBS, no calcium, no magnesium

  2. Wash Buffer (WB)

    10 g BSA (final concentration 2%)

    2 ml EDTA from 0.5 M stock (final concentration 2 mM)

    Add 500 ml 1× DPBS, no calcium, no magnesium

    Filter through 0.45 μm pores

  3. Blocking Buffer (BB) (keep sterile)

    15 g BSA (final concentration 3%)

    5 ml porcine skin gelatin stock (final concentration 0.2%)

    Add 50 ml 1× DPBS, no calcium, no magnesium

    Filter through 0.45 μm pores

  4. Porcine skin gelatin stock 2%

    2 g porcine skin gelatin

    100 ml ddH2O

  5. 0.6 M KCl (keep ice-cold)

    37.28 g KCl granular

    1,000 ml ddH2O

    Filter through 0.45 μm pores

  6. Neutrophils complete medium (keep sterile)

    5 ml L-glutamine from 200 mM stock (final concentration 2 mM)

    500 ml RPMI-1640 without L-glutamine


Authors would like to thank Dr. Mariana Kaplan (NIAMS) and Dr. Phil McCoy and the NHLBI Flow Cytometry Core, for their excellent scientific and technical advice; Jim Nichols and Darlene Allen for assisting with the recruitment of blood donors and Dr. Laxminath Tumburu for his help with the figures. This work was supported by the Intramural Research Program of the National Heart, Lungs, and Blood Institute, NIH (to SLT).

Competing interests

The authors declare no competing financial or non-financial interests.


Informed consent was obtained from all subjects who provided blood samples enrolled in the study protocol NCT0004799, approved by the NHLBI Institutional Review Board.


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[摘要]中性粒细胞胞外陷阱(NETs)的形成被认为在感染和传播无菌炎症中起关键作用。组蛋白瓜氨酸化是NETs形成中必不可少的早期步骤,可在形成标志性的细胞外DNA支架链之前检测到。除了经典的显微镜方法外,还在研究和临床目的开发用于NETs及其检测的新技术。NET的经典显微镜研究具有主观性,低通量和半定量性,并且捕获早期步骤的能力有限。我们开发了这种新颖的成像流式细胞仪 (IFC)方法,专门识别和量化组蛋白H4的瓜氨酸化作为NETs标记及其与核和细胞水平其他改变的关系。这些包括核解聚和超缩合,多分叶状核与1-叶核和细胞膜损伤。在形成特定的细胞外DNA支架链之前,可以在用NETs诱导剂进行不同的治疗期后对NETs标记物进行定量。由于这些高通量基于图像的细胞分析功能可以通过严格的统计来执行,因此该协议适用于实验和临床应用以及作为生物标志物的NETosis的临床评估。


除了经典的广泛使用的免疫荧光显微镜技术外,其他的NETs检测和定量方法也正在开发中。这些包括使用共焦显微镜,以提供有关NETs结构的信息(Santos等人,2018),基于ELISA的测定法来定量人血浆中的瓜氨酸化组蛋白3(H3cit)(Thalin等人,2017),高使用人中性粒细胞中的膜可渗透和不可渗透核染料进行通量实时检测NETosis和凋亡相关的核变化(Gupta等人,2018),使用微流控技术对人全血中游离染色质(主要与弹性蛋白酶相关)进行定量(Muldur等人,2018)。多项研究使用常规流式细胞仪监测人和小鼠中的NETs成分(H3cit,MPO和细胞外DNA)(Gavillet等,2015; Masuda等,2017; Lee等,2018; Zharkova等,2019) )。尽管该技术可以进行高通量分析,但在探针选择,样品制备以及门控影响最终结论的方式等方面也确实存在明显的局限性(Manda-Handzlik等,2016; Masuda等,2017)。 )。另一方面,尽管成像流式细胞术(IFC)确实可以进行高通量分析,但它还结合了常规流式细胞术和荧光显微镜的可视化和分析功能(Basiji,2016)。利用IFC研究了NETs相关的形态核变化(即染色质解聚和DNA谱线)以及MPO是否与核室共存以及在何种程度上共存(Zhao等人,2015; Ginley等人,2017; Pelletier等人)等人,2017)。因此,目前采用不同的标记物和不同的方法在体外检测和定量NETs 。长期以来,PAD4介导的组蛋白瓜氨酸化一直被认为是NETs形成的标志,因此是遵循的理想标志物。然而,最近的报告主要在小鼠模型中表明,PAD4介导的组蛋白3(H3cit)的瓜氨酸化是依赖于刺激的(Neeli和Radic,2013; Claushuis等,2018; Guiducci等,2018; Liang等人,2018),因此该站点作为NETs标记的用途可能受到限制。

在这里,我们详细描述了一种新颖的IFC协议,该协议允许在将DNA和细胞质蛋白链释放到细胞外空间之前,对整个中性粒细胞中作为NETs标记的组蛋白4(H4cit3)的瓜氨酸化进行特异性检测和定量。其他分析参数看核和细胞形态学变化(核解聚和supercondensation ,多呈分叶状细胞核和细胞膜损伤)可以带来额外的信息息对所分析的嗜中性白细胞群体的行为。为了建立我们的方法,我们确定了使用NETs激动剂治疗5个不同时间段(2分钟至60分钟)的健康人类嗜中性粒细胞的反应:药理学诱导剂,PMA和钙离子载体(组蛋白瓜氨酸化的著名诱导剂,以及我们的实验条件(阳性对照),Hemin (在溶血条件下存在的诱导剂)和LPS和IL-8(与传染性病原体相关的诱导剂)。我们使用了体外荧光显微镜检查,以确认健康的中性粒细胞中经IFC测试使用的刺激处理后DNA弹性蛋白酶-MPO链的形成(Barbu等人,2020年)。我们进一步在健康供体和稳态下具有镰状细胞疾病患者的未经治疗和经过Hemin治疗的中性粒细胞中验证了该技术(Barbu等,2019)。


此protoco升有4个主要部分组成:一步骤嗜中性粒细胞的纯化,诱导NETosis ,特异性染色,IFC采集和分析(如图中所强调小号1A和1C),与需要3天,最多一周的前3个步骤完成所有步骤。

图1.通过成像流式细胞术特异性检测NETosis标记的主要步骤的日常描述。A.使用Polymorphprep培养基从全血中一步纯化多形核(PMN)细胞;用这种方法,中性粒细胞的纯度应始终高于80%。总处理时间:90分钟。B.使用Polymorphprep梯度介质正确分离全血的示例。C.多形核细胞治疗和染色程序的详细描述。不包括一时间推荐的中性粒细胞刺激一步研究人员应该根据自己experimen选择合适的治疗时间TAL目的。每个洗涤步骤最多可能需要20分钟才能完成,具体取决于所处理样品的数量。在计算每天完成所有步骤所需的总时间时,应考虑该时间。❖短暂的最后清洗,然后将样品保存在4 ℃。总的刺激和š泰宁处理时间为1天:4+小时。

关键字:中性粒胞外菌网的探测, 定量中性粒胞外菌网, 成像流式细胞术, NETosis, 组蛋白H4瓜氨酸化, 核解凝

识破,Lav的ë的nDer ,10.0 V,16 × 100毫米,塑料管BD Hemograd封闭(BD,目录号:BD366643)
聚丙烯锥形管,50毫升(Fisher Scientific,Corning,目录号:07-203-510)
离心管,5 ml,锥形底部,无菌(Benchmark Scientific,目录号:C1005-T5-ST)
1.5 ml离心管,混合霓虹色(美国科学公司,目录号:1415-1448)
快速流动的无菌一次性过滤单元与PES膜,容量500毫升,孔径0.45微米物(Thermo Scientific的Nalgene,目录号:09-740-63E)
Polymorphprep梯度培养基(Cosmo Bio USA,目录号:AXS-1114683)

不含L-谷氨酰胺的RPMI-1640(Lonza ,目录号:12-167F)
L-谷氨酰胺200 mM (Thermo Fisher,目录号:25030-081)
注意:将等分试样一次使用,并储存在-80 °C下。在37 °C的水浴中解冻以溶解白色沉淀物,然后添加到RPMI中以制成完整的中性粒细胞培养基。

超纯0.5 M EDTA pH 8.0(Thermo Fisher ,Invitrogen,目录号:15575020)
DPBS 1 × ,无氯化钙,无氯化镁(Gibco ,目录号141190-136)
牛血清白蛋白,热激分数,无蛋白酶,pH 7 .0 ,≥98%(Millipore Sigma,目录号A3294)
二甲亚砜(DMSO),≥ 99.5%(GC),植物细胞培养物测试(Sigma-Aldrich公司,目录号:D4540)
牛血红素,≥90 %(Millipore Sigma,目录号:9039)
注意:准备DMSO中的40 mM储备液,以单次使用的馏分等分并在-20 °C下储存。准备工作溶液时要充分混合。添加带有测试样本的仅DMSO控件。

PMA,用于分子生物学应用,≥99 %,HPLC(Millipore Sigma,目录号:P1585)
注意:在DMSO中准备储备溶液,然后将单次使用的等分试样在-20 °C下冷冻。

LPS-EB,超纯,大肠杆菌0111:B4(InvivoGen Corporation制,目录号:TLR11的-3pelps)
注意:准备一份ddH 2 O的储备溶液,等分并在-20 °C冷冻。将解冻冻结周期限制为三个。

重组人IL-8(CXCL7,77aa)(Peprotech ,目录号:200-08)
钙离子载体A23187 ≥ 98%(TLC),粉末(Millipore公司Sigma,目录号:C7522)
抗人类CD66b-PE,克隆G10F5(Biolegend ,目录号305106)
兔多克隆抗组蛋白H4,瓜氨酸3(H4cit3,Millipore Sigma,目录号:07-596)(请参见注释)
山羊抗兔IgG (H + L)二抗,DyLight 680(Thermo Fisher,目录号:35568)(请参阅注释)
MPO多克隆抗体,已偶联AlexaFluor 594(Bioss抗体,目录号:bs-4943R-A594)
Hoechst 33342(BD Pharmingen ,目录号:561908)
来自猪皮的明胶,粉末,A型,适用于细胞培养(Millipore Sigma,目录号:G1890)
注意:准备2%的库存,并将其在4 °C下保存最多6个月。仅在无菌罩中打开。监视细菌或真菌污染的迹象。

BD Cytofix / Cytoperm试剂盒(BD Biosciences,目录号:554722)。在此协议中仅使用Cytofix / Cytoperm解决方案
Paraformaldeh ý德8%瓦特工作会有溶液(见配方)
0.6 M KCl (请参阅食谱)


W¯¯亚特浴在37 ℃下
LabGard ® ES,II级,A2型,生物安全柜(NuAire ,普利茅斯,MN)
台式离心机,带有可旋转的桶形转子(贝克曼库尔特,型号:Allegra X-14R)
离心机(Thermo Fisher Scientific,型号:Sorvall ST 16R)
CO 2培养箱(Thermo Fisher Scientific,型号:Heracell VIOS 160i)
Amnis ImageStream Mark II成像流式细胞仪(Luminex Corporation,美国德克萨斯州奥斯汀)


Amnis INSPIRE(Luminex公司,美国德克萨斯州奥斯汀)进行数据采集
Amnis IDEAS(Luminex公司,美国德克萨斯州奥斯汀)进行数据分析,可在公司网站上下载



选择一种高产率的一步中性白细胞纯化方法,因为该NETs检测方案需要相对大量的中性白细胞(每个样品2 × 10 6 ),且背景活化最少。
离心步骤必须在摆桶式转子中进行,因为必须在试管中间,血液与Polymorphprep介质之间的界面处达到500 × g的速度。

将离心机的转子和适配器平衡至室温(18 °C至22 °C )。将“加速度”设置为一;将“减速度”设置为零。将离心力设置为500 × g 30分钟。
收集中性粒细胞环并轻轻重悬于50 ml冰冷的DPBS中。在4 °C下以400 × g离心10分钟,最大加速度和减速度设置为。除去并丢弃含血小板的上清液。
轻弹沉淀以重悬,然后加入3 ml冰冷的无菌水,并通过轻轻上下吹打几次确保沉淀完全重悬。30 s后,用6 ml无菌冰冷的0.6 M KCl恢复渗透压。充满冰冷的DPBS,在330 × g下离心,在4°C下7分钟。
弃去上清液,然后将沉淀重新悬浮在室温下平衡的中性粒细胞完全培养基(RPMI补充2 mM L-谷氨酰胺)中,浓度为4 × 10 6细胞/ ml。


对于每个实验,在1 × DPBS中准备新鲜的8%PFA稀释液,并将其在黑暗中保持阴凉直到需要使用(在下一步中将其进一步稀释至4%的最终浓度。请勿储存用于第二次使用)。
如果如此选择,下面的所有步骤都可以直接在1.5 ml Eppendorf管中进行。但是,通过移液将PFA剧烈混合到细胞悬液中会导致细胞飞溅和丢失。使用5毫升试管可提供更多空间,以进行充分混合并防止细胞损失。

添加纯化的嗜中性粒细胞,2 × 10 6细胞(在500 μ升完整嗜中性粒细胞的培养基)在5毫升的无菌管中,并允许纯化嗜中性粒细胞在室温下平衡。
将试管转移到37°C的5%CO 2的培养箱中,并让它们静置至少30分钟而不关闭瓶盖。
添加100  μ完全RPMI的l为未处理的对照或RPMI含有选择的母语诱导物,轻轻所有管和回流管中的相同的次数混合以培养箱为期望的时间段。在此孵育期间,请勿关闭管盖。
从培养箱中并停止反应通过除去dding 600 μ升新鲜制备的8%PFA的在1 × DPBS为4%PFA的最终浓度,通过移液混合至少5倍,并允许在室温下静置至少30分钟,盖上铝箔。
加入1ml洗涤缓冲液(WB在室温和离心机)在1 ,120 ×克5分钟,在4℃下。将含有PFA的上清液丢弃到指定的化学废物中。为确保除去PFA,请再次重复此洗涤步骤。
轻轻重悬浮沉淀用500 μ升WB的,并将其传输到1.5毫升的Eppendorf管中用于随后的染色步骤。
重复5-ml管的洗涤与另一个5 00 μ升WB的,以确保所有细胞已被收集用于染色。


通过用高于和低于供应商推荐的一系列稀释液进行滴定来确定染色板中使用的所有抗体的最佳工作稀释度(例如,对于建议的1:1:1稀释度,应最多添加5种不同的稀释度)经过测试 – 1:250;1:500、1:1,000、1:2,000、1:3,000)。这是必需的,因为不同ImageStream细胞仪上的激光器可能具有不同的功率。

                 在1.5 ml的Eppendorf管中,将固定并彻底洗涤的细胞重悬于100μl含CD66b-PE的2%BSA中,用移液器剧烈混合至少5次,并在室温下于黑暗中孵育20分钟。
                 洗涤步骤:加入500 μ升WB和离心机在1120 ×克5分钟,在4℃下。除去上清液,并重复清洗两次。
                 重新悬浮在300洗涤的沉淀μ升BD的CYTOFIX / Cytoperm ,通过上下抽吸至少5倍,孵育15分钟,在室温下混合,在黑暗中。
在100重悬μ升含有2%BSA的抗组蛋白H4瓜氨酸3(H4cit3)初级抗体在最佳确定稀释。上下吸移至少10次,并在4 °C下孵育过夜。

在确定的最佳稀释度下,将微球重悬于100μl含2%BSA的二抗DyLight 680中,并与抗MPO- AlexaFluor 594偶联。至少用移液器上下吸移10次,在黑暗中于室温下孵育30分钟。
在200重悬μ升2%的含有稀释的Hoechst由供应商(:1000 1)所建议的BSA。通过移液彻底混合,并在黑暗中于室温下孵育15分钟。
加入500 μ升WB和离心机在1120 ×克5分钟,在4℃下。除去上清液。
在50重悬μ升WB为的ImageStream (成像流式细胞仪)获取(见注D和E段)。


在“照明”选项卡下,基于荧光染料面板打开785激光器(侧面散射激光器– SSC)和所有其他要在实验中使用的激光器(可在屏幕上找到显示激光激发波长及其相应染料的图表)。公司的网站)。
在“放大倍数”部分下,将放大倍数调整为60 × 。
在“文件获取”下,创建一个新文件夹以保存获取的文件(扩展名为。rif –原始图像文件)。
加载具有所有颜色染色的样品的d预期产生最亮的信号,用于所使用的所有染料(用PMA作为示例-这里的理性是确保正荧光信号在范围内,不会被饱和的通道) 。
Ñ OTE:当设置信道特定的激光功率,激光的最佳电压是一个不饱和的检测器(小于4096个像素所生成的图像中),并且也促进了阴性和阳性群体之间有明显的区别。每个已使用通道均应具有“原始最大像素”功能。此功能提供了输入蒙版中包含的像素的最高价值。

以产生补偿矩阵文件,启动上激发补偿向导,并获取至少1 ,从每个补偿控制管000的事件。
注意:Amnis ImageStream网站上的客户门户网站提供了有关如何使用INSPIRE(购置软件)中的向导运行和验证矩阵的详细说明。补偿矩阵必须应用于每个获取的。rif文件,首次在IDEAS软件中打开以进行分析(从n开始,当使用INSPIRE获取该文件时还应用了补偿矩阵)。溢出补偿可以在数据文件的采集或采集后进行。如果获取的文件的停止栅极基于荧光信号,例如,获取基于对CD66b 5000个阳性细胞,然后它是可取的上INSPIRE执行补偿(使用补偿向导),以确保荧光信号被校正用于其他荧光通道的溢出。

创建亮区面积(BF)与侧向散射(SSC)通道强度的点图,然后单击点(群体)并评估图库中显示的对应图像,可以找到单个单元格和边界决定。在此协议中,对于纯化的中性粒细胞,单个细胞的门的面积应在50 µm 2至200 µm 2的范围内。这应该排除大部分的碎片,小颗粒(在底部和左侧)和骨料(略右上-请参考˚F igure 2A)。
注:d UE,以中性粒细胞的性质,我们认为这将是更容易识别汗衫通过使用面积与SSC的; 但是,也可以使用BF的面积与纵横比(AR)来确定单个单元格。长宽比是宽度与高度的比率,应将单个事件定位在距离AR接近1的事件处。






创建BF图像(M01 –默认遮罩)与侧面散射(SSC –通道6)的面积的点图。

通过使用明场中的聚焦质量功能Gradient RMS(均方根)来选择聚焦的单元格。
创建粒细胞特异性标志物CD66b与Hoechst和门双重阳性事件(CD66b + Hoechst +)信号强度的点图,以进行进一步分析。
在CD66b + Hoechst +门中,为H4cit3信号的强度创建直方图,并评估该标记的H4cit3 +细胞百分比和/或均值/中值荧光(图2B)。

在核通道(Hoechst公司)的CD66b +的Hoechst +栅极创建“区”的一个点图相对于特定纹理特征“明亮细节Intensity_R3” (BDI_R3)以识别与嗜中性粒细胞解聚从那些在CD66b +的Hoechst +栅极正常核细胞核(图2C)。
注意:BDI是一项功能,可以计算图像中被遮罩区域内的局部亮点的强度(此协议以及此协议中使用的其他IDEAS功能的描述可以在可下载的IDEAS ImageStream Analysis Software用户手册中找到)。

图2.使用IDEAS软件识别中性粒细胞及其核NETosis标记的门控策略。A.用明场区域(BF – X轴)与通道6(SSC – Y轴)上的侧向散射强度(左图)的两参数点图识别的单个单元格。通过使用Brightfield (中间面板)中的Gradient RMS(均方根)来选择聚焦的单重态细胞。嗜中性粒细胞(CD66b + Hoechst +,草莓红)在通道7的Hoechst荧光(X轴)和通道3的CD66b(Y轴)(右图)的两参数荧光点图中由单线态聚焦细胞鉴定。B.代表性直方图显示CD66b + Hoechst +门中H4cit3 hi细胞的百分比和H4cit3中值荧光强度(MFI)。在右侧面板的嗜中性粒细胞在RPMI不处理(红色)或用共治疗20 ü中号氯化血红素15分钟(绿色)。C.解聚核(紫色)和正常核(绿色)用识别d OT-情节核的面积(吗啉Hoechst公司的迟缓掩模功能染色上通道7)相对于Hoechst公司的亮度细节强度R3从染色的核的(例子这两个门位于面板的右侧)。Ñ体中性不处理(左)或具有共治疗20 μ中号氯化血红素60分钟(右)。显示了具有缩合核的细胞的百分比。比例尺= 7μm 。


使用核染料通道上自定义的“水平集”蒙版和“ LobeCount ”功能检测具有多叶核的细胞百分比的减少,并分别量化具有可变叶数(1叶到4叶)的细胞(图3B)。


图3. IDEAS软件分析功能可识别和量化其他核和细胞改变。A. DNA(Hoechst –通道7)和MPO(AF-594 –通道4)的相似性分析功能表明,在具有正常核(绿色)和缩合核(紫色)的嗜中性粒细胞中,核区与I型颗粒共定位。包括具有低(< 1)和高(3)相似度分数的细胞示例,分别指示低和高DNA-MPO共定位(其特定位点在图中的位置以浅绿色表示)。B.核瓣计数功能可根据核图像识别核瓣数目的变化,并量化每个类别中的细胞数目。包括具有1个叶或多裂核的嗜中性粒细胞的代表性图像。C. X轴上核面积的点图(Hoechst染色的形态学掩模)与1个叶核门中整个细胞面积的核比例之比确定了低核面积的超浓缩核,因此除以核素后的比例很低。整个细胞区域。呈现了具有超浓缩核的细胞的代表性图像。D.具有受损膜的中性粒细胞(亮绿色)通过通道6(X轴)与明场调制(Y轴)上标准偏差侧向散射的两参数点图来区分。两项功能均获得高分表示复杂和有纹理的对象。包括两个闸门内细胞的代表性图像。(BF =明场; H =赫斯特)。比例尺= 7μm 。

建立所有分析参数后,保存文件。对于每个。rif采集文件,在分析步骤中创建了两个附加文件(.daf和.cif )。使用 。daf进行分析。不要删除。cif一个(这是所有分析数据的存储位置)。
在里面 。daf分析文件使用“报告”为目标亚群中所有感兴趣的参数定义统计报告,然后使用“生成统计报告”将其应用于所有分析的样本文件(图4)。



通过运行带有细胞和二抗DyLight 680抗体但无H4cit3一抗的对照管,确认二抗(DyLight 680)没有非特异性结合。
使用自定义遮罩和IDEAS软件进行数据分析,因为它们可以更准确地表示关注区域。通过确认掩膜确实掩盖了感兴趣的区域(即,没有丢失目标区域的一部分,或者相反,掩盖了什么区域之外的“重影”区域)来验证自定义掩膜的总细胞面积和细胞核与功能相关)(Dominical et al。,2017)。
请注意,该协议允许在将DNA和细胞质蛋白串释放到细胞外空间之前,在整个嗜中性粒细胞中鉴定NETosis标记。这个细节对于决定NETs诱导剂对刺激做出反应的峰值细胞数的挑战时间至关重要,并且可以通过ImageStream细胞仪获取。长时间的刺激可能会导致假阴性结果,原因是细胞已经溶解并释放了它们的内含物,因此对于流式细胞仪不再“可见”。这种情况可以通过独立评估来确认:例如,通过在染色管中观察到的沉淀物的大小,以及与之相比,通过流式细胞仪在受激管中主动捕获的CD66b + Hoechst +门中的事件数减少了。未经处理的对照。
在IDEAS软件中,打开一个。在daf扩展文件中,选择一个Hoechst + CD66b +双阳性细胞。
单击“关闭”关闭mas k窗口。
选择“ /”(正斜杠)以计算比率。


8%低聚甲醛(PFA)  工作溶液
2.5 ml PFA 32%库存

加入10毫升1 × DPBS,无钙,无镁



加入500毫升1 × DPBS,无钙,无镁




加入50毫升1 × DPBS,无钙,无镁

过滤0.45 μ米孔隙


100毫升ddH 2 O

0.6 M KCl (保持冰冷)
37.28 g氯化钾颗粒

1,000毫升ddH 2 O


从200 mM储备液中提取5 ml L-谷氨酰胺(终浓度2 mM )

500 ml不含L-谷氨酰胺的RPMI-1640


作者要感谢医生。马里亚纳卡普兰(NIAMS)和博士。菲尔·麦科伊(Phil McCoy)和NHLBI流式细胞仪核心,为其提供了出色的科学和技术建议;吉姆·尼科尔斯和达琳·艾伦与献血者和博士的招聘协助。Laxminath Tumburu为他提供了帮助。这项工作得到了美国国立卫生研究院国家心脏,肺和血液研究所(至SLT)的壁内研究计划的支持。






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引用:Barbu, E. A., Dominical, V. M., Mendelsohn, L. and Thein, S. L. (2021). An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation. Bio-protocol 11(4): e3927. DOI: 10.21769/BioProtoc.3927.

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