Induction and Quantification of NETosis

Carmelo Carmona‐Rivera1, Mariana J. Kaplan1

1 Systemic Autoimmunity Branch, Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.41
DOI:  10.1002/cpim.16
Online Posting Date:  November, 2016
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Neutrophil extracellular traps (NET) are web‐like structures composed of nuclear material and neutrophil granular proteins that are released to the extracellular space after specific microbial or sterile inflammatory stimuli. NETosis is a specific type of cell death that ensues when neutrophils extrude NETs. It is important to develop validated standards to induce and quantify NETs in order to accurately compare results between laboratories and advance the understanding of this biological process and its implications in health and disease. This unit presents methods for isolating human neutrophils from peripheral blood and inducing NETs through various types of stimulation that rely on the production of reactive oxygen species from NADPH oxidase or mitochondria. Alternative methods are also described for low‐ and high‐throughput quantification of NETs. © 2016 by John Wiley & Sons, Inc.

Keywords: flow cytometry; immunofluorescence; neutrophils; neutrophil extracellular traps

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Table of Contents

  • Introduction
  • Basic Protocol 1: Induction of NETosis and Quantification by Immunofluorescence
  • Alternate Protocol 1: Induction of NETosis and Quantification by Multispectral Imaging Flow Cytometry
  • Alternate Protocol 2: Induction of NETosis and Quantification by Plate Assay
  • Support Protocol 1: Isolation of Human Neutrophils Using Polymorphprep
  • Support Protocol 2: Isolation of Human Neutrophils Using the EasySep Human Neutrophil Enrichment Kit
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Induction of NETosis and Quantification by Immunofluorescence

  • Isolated purified neutrophils (e.g., unit 7.23 or protocol 4 or 2 below)
  • RPMI‐1640 medium without phenol red
  • Phorbol 12‐myristate 13‐acetate (PMA; Sigma, cat. no. P1565)
  • Calcium ionophore A23187 (Sigma, cat. no. C7522)
  • 4% (w/v) PFA (see recipe)
  • PBS, pH 7.4 ( appendix 2A)
  • Blocking buffer (see recipe)
  • Anti‐myeloperoxidase (Dako, cat. no. A0398) or anti‐elastase antibody (Abcam, cat. no. ab21595)
  • Secondary antibody (e.g., for rabbit primary antibody, use goat anti‐rabbit IgG Alexa Fluor 555)
  • Hoechst 33342
  • ProLong Gold antifade mounting medium
  • 24‐well plate
  • Poly‐L‐lysine‐coated 12‐mm round glass coverslips
  • 1.5‐ml microcentrifuge tubes
  • Humidified incubator (37°C, 5% CO 2)
  • Curved Swiss Jewelers forceps
  • 75 × 25 × 1–mm microscope slides
  • Epifluorescence or confocal microscope equipped with 40× objective and filters to detect excitation/emission at 350/461 nm (Hoechst) and 555/565 nm (Alexa Fluor 555)
  • Imaging software (e.g., Photoshop or ImageJ)

Alternate Protocol 1: Induction of NETosis and Quantification by Multispectral Imaging Flow Cytometry

  Additional Materials (also see protocol 1Basic Protocol)
  • FITC‐coupled anti‐MPO antibody (Abcam, cat. no. ab11729)
  • 2% (w/v) paraformaldehyde
  • Multispectral Imaging Flow Cytometer (Amnis)

Alternate Protocol 2: Induction of NETosis and Quantification by Plate Assay

  Additional Materials (also see protocol 1Basic Protocol)DNA dye(s):
  • Sytox Green (cell impermeable; Thermo Fisher, cat. no. S7020)
  • Sytox Orange (cell impermeable; Thermo Fisher, cat. no. S34861) plus PicoGreen (cell permeable; Thermo Fisher, cat. no. P7589)
  • Laminar flow hood
  • 96‐well black plate
  • Microplate reader equipped with filter to detect excitation/emission at 488/520 nm

Support Protocol 1: Isolation of Human Neutrophils Using Polymorphprep

  • Polymorphprep gradient medium (Cosmo Bio USA, cat. no. AXS‐1114683)
  • 15‐ml human blood collected in EDTA‐treated tube
  • PBS ( appendix 2A)
  • RPMI‐1640 medium without phenol red
  • Laminar flow hood
  • 15‐ and 50‐ml conical tubes
  • Hemacytometer

Support Protocol 2: Isolation of Human Neutrophils Using the EasySep Human Neutrophil Enrichment Kit

  • HetaSep (red blood cell sedimentation reagent; Stemcell Technologies, cat. no. 07806)
  • 10‐ml human blood collected in anti‐coagulant‐treated tube
  • Supplemented PBS (see recipe)
  • EasySep Human Neutrophil Enrichment Kit (Stemcell Technologies, cat no. 19257) containing enrichment cocktail and magnetic particles
  • Laminar flow hood
  • 15‐ and 50‐ml conical tubes
  • EasySep Magnet
  • Hemocytometer
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Literature Cited

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