In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

Published: November 30, 2023

Abstract

Source: Zhang, Y., et al. In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages. J. Vis. Exp. (2019).

This video demonstrates in vitro stimulation of macrophage extracellular traps (METs) in human monocyte-derive macrophages using different inflammatory stimuli such as TNF-alpha, PMA, and HOCL. The video also demonstrates the method of SYTOX dye staining of METs for visualization.

Protocol

1. Polarization of HMDM

  1. Under sterile conditions, prepare the M1 priming media by adding interferon γ (IFNγ; 20 ng/mL) and lipopolysaccharide (LPS; 1 μg/mL) to the complete RPMI-1640 culture media. Prepare the M2 priming media by adding interleukin 4 (IL-4; 20 ng/mL) to the complete RPMI-1640 culture media.
  2. Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and cultured as described in section 1.
  3. Carefully wash the wells containing the cells 3x with sterile PBS (pre-warmed to 37 °C), using 1 mL aliquots of PBS.
  4. Add 1 mL of either the M1 or M2 priming media to each well containing the HMDM (whichever is appropriate for the experiment).
  5. Incubate the cells for 48 h at 37 °C in the presence of 5% CO2 in a cell incubator.

2. Stimulation of HMDM to induce MET Release

  1. Under sterile conditions, prepare the culture media containing different stimulators of macrophage extracellular traps MET release (whichever is appropriate for the experiment) to the complete RPMI-1640 media: PMA (25 nM), human recombinant TNFα (25 ng/mL), or human recombinant IL-8 (50 ng/mL).
  2. For experiments with hypochlorous acid, HOCl stimulation, prepare HOCl (200 μM) in HBSS (pre-warmed to 37 °C), immediately before the addition to the cells. Ensure that the HOCl is not prepared in complete cell media.
    NOTE: The concentration of the stock solution of HOCl is quantified by measuring the UV absorbance of the solution at 292 nm and pH = 116 and using an extinction coefficient of 350 M-1cm-1.
  3. After the polarization treatment described in section 2, aspirate the cell media from each well and carefully wash the cells 3x with 1 mL aliquots of either: sterile PBS (for PMA, TNFα and IL-8) or HBSS (for HOCl), which have been pre-warmed to 37 °C.
  4. For experiments with PMA, TNFα, or IL-8: add 1 mL of the complete media containing PMA, TNFα, or IL-8 after removing the PBS in the final washing step.
  5. For experiments with TNFα, incubate the cells for 6 h at 37 °C in the presence of 5% CO2 in a cell incubator. For experiments with PMA and IL-8, incubate the cells for 24 h at 37 °C in the presence of 5% CO2 in a cell incubator.
  6. For experiments with HOCl, add 1 mL of HOCl in HBSS after removing the HBSS in the final washing step. Then, incubate the cells for 15 min at 37 °C in the presence of 5% CO2 in a cell incubator.
    1. Carefully aspirate the cell supernatant and wash the cells 3x with 1 mL aliquots of HBSS as described in step 2.3.
    2. After removing the HBSS from the final wash step, add 1 mL of complete RPMI-1640 culture media. Then, incubate the cells for 24 h at 37 °C in the presence of 5% CO2 in a cell incubator.

3. Visualization of MET in Live Cell Culture

  1. Prepare SYTOX green dye in HBSS at a concentration of 40 μM.
  2. At the end of treatments described in section 2 to induce MET release, directly add 25 μL of 40 μM of the dye to each well containing HMDM.
  3. Incubate cells at room temperature (RT) for 5 minutes in the dark.
  4. Place the HMDM in tissue culture wells on the microscope stage of an inverted fluorescent microscope for imaging.
  5. Microscope procedures
    1. Turn on a broad-spectrum fluorescent light source, brightfield light source, and inverted microscope installed with a high-resolution color digital camera (see Table of Materials).
    2. Rotate the filter wheel to the "number 2" position for green fluorescence (excitation = 504 nm, emission = 523 nm) for imaging of the green stained samples contained within the tissue culture wells.
    3. Using the 5x objective, focus the image with the coarse focus, then the fine focus knobs on the microscope, until the image appears sharp, clear, and focused when viewed through the microscope eyepiece.
    4. Switch the microscope to the camera mode.
    5. Start the associated software.
    6. Select the Capture tab on the software.
    7. Click the Play button to preview the image and adjust the fine focus knob on the microscope until the image appears sharp, clear, and focused in the software preview window.
    8. Click the Capture button.
      NOTE: The captured image will automatically be displayed in the accompanying software.
    9. Within the software, click File | Save as the required image file type.
    10. On the microscope, rotate the filter wheel to the "number 5" position for brightfield imaging and repeat steps 3.5.2–3.5.9 to obtain the corresponding brightfield image.
    11. Repeat the steps 3.5.2–3.5.10 as necessary for subsequent image acquisition.

Disclosures

The authors have nothing to disclose.

Materials

120Q broad spectrum fluorescent light source EXFO Photonic Solutions, Toronto, Canada x-cite series
Corning CellBIND Multiple Well Plate (12 wells) Sigma-Aldrich CLS3336 For cell culture
Hypochlorous acid (HOCl) Sigma-Aldrich 320331 For MET stimulation
Interferon gamma Thermo-Fisher PMC4031 For M1 priming
Interleukin 4 Integrated Sciences rhil-4 For M2 priming
Interleukin 8 Miltenyl Biotec 130-093-943 For MET stimulation
L-Glutamine Sigma-Aldrich 59202C Added to culture media
Olympus IX71 inverted microscope Olympus, Tokyo, Japan
Phorbol 12- myristate 13-acetate (PMA) Sigma-Aldrich P8139 For MET stimulation
Phosphate buffered saline (PBS) Sigma-Aldrich D5652 For washing steps
RPMI-1640 media Sigma-Aldrich R8758 For cell culture
SYTOX green Life Technologies S7020 For MET visulaization
TH4-200 brightfield light source Olympus, Tokyo, Japan x-cite series
Tumor necrosis factor alpha Lonza 300-01A-50 For MET stimulation

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Cite This Article
In Vitro Stimulation and Visualization of Macrophage Extracellular Traps. J. Vis. Exp. (Pending Publication), e21788, doi: (2023).

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