Immunology and Infection
Published: August 24th, 2018
We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.
In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.
Neutrophils are short-lived granulocytes responsible for the initial defense against invading pathogens. Neutrophils, recruited to the site of infection, eliminate microorganisms by phagocytosis, degranulation, and generation of reactive oxygen species (ROS)1. In the presence of bacteria or endotoxins, neutrophils release neutrophil extracellular traps (NETs), composed of extracellular chromatin decorated with histones and granular proteins like elastase and myeloperoxidase (MPO)2. Although NETs have indispensable antimicrobial properties, increasing experimental and clinical evidence suggests that overzealous NET format....
All methods described here were approved by the Institutional Animal Care and Use Committee at the University of California, Davis (protocol number: 18338).
1. Blood Collection
Using this protocol of live cell imaging, investigators can observe the nuclear morphology, plasma membrane integrity and presence of cfDNA in living neutrophils. A cell impermeant nuclear dye stains nuclei acids red in cells with damaged cell membranes. Another cell-permeant dye, labels intracellular nucleic acids in live cells with intact plasma membranes. All intact neutrophils, regardless of their treatments, should appear green and exhibit the characteristic lobulated nuclei at 0 to .......
We present here a protocol to observe the changes in nuclei conformation and cfDNA release in living canine neutrophils using both a cell permeant dye and a cell impermeant dye. The main advantage of this assay is that it allows for real-time detection of NET formation by high-resolution microscopy in live neutrophils without cell fixation, therefore, providing a simple and valuable tool for observing in vitro NET formation. Since this assay does not require antibodies for the detection of NET components, it is .......
The corresponding author was funded by the Morris Animal Foundation (D15CA-907). The study was supported by funds from the University of California, Davis, Center for Equine Health and Center for Companion Animal Health (2016-24-F). The authors would like to acknowledge Geena Ng for her assistance with the figures and Nghi Nguyen for her assistance with the video.....
|Dextran from Leuconostoc spp.
|Molecular weight 450,000 – 650,000
|GE Life Sciences
|Dulbecco’s Phosphate-Buffered Saline
|With divalent cations
|Dulbecco’s Phosphate-Buffered Saline
|Without divalent cations
|E. coli O55:B5
|SYTOX Orange Fluorescent Nucleic Acid Stain
|5 mM in DMSO; stains in cells with permeable membranes
|SYTO Green 16 Fluorescent Nucleic Acid Stain
|1 mM in DMSO; stains in cells with intact membranes
|Poly-D-Lysine coated coverslips
|12 mm diameter
|Anti-citrullinated histone H3 antibody
|Unconjugated goat anti-rabbit Fab fragments
|Specificity: IgG (H+L)
|Anti- Myeloperoxidase antibody
|Life Technologies Corporation
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