In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Draw 10 mL of blood from either the cephalic or jugular vein using a 21 G needle by syringe aspiration.
2. To avoid excessive shear stress, remove the needle from the syringe before transferring blood into tube(s) containing sodium heparin (75 USP). Gently invert the tubes a few times to ensure adequate mixing of anticoagulant and blood. Check for evidence of clots or red cell aggregates before placing the samples on ice.

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dextran from Leuconostoc spp.	Sigma	31392	Molecular weight 450,000 – 650,000
Ficoll-Paque PLUS	GE Life Sciences	17144002
Dulbecco’s Phosphate-Buffered Saline	ThermoFisher Scientific	A1285801	With divalent cations
Dulbecco’s Phosphate-Buffered Saline	ThermoFisher Scientific	14190136	Without divalent cations
E. coli O55:B5	InvivoGen
SYTOX Orange Fluorescent Nucleic Acid Stain	ThermoFisher Scientific	S11368	5 mM in DMSO; stains in cells with permeable membranes
SYTO Green 16 Fluorescent Nucleic Acid Stain	ThermoFisher Scientific	S7578	1 mM in DMSO; stains in cells with intact membranes
Surface-Amps NP-40	Pierce	28324
Poly-D-Lysine coated coverslips	neuVitro	H-12-pdl	12 mm diameter
Anti-citrullinated histone H3 antibody	Abcam	Ab5103
Unconjugated goat anti-rabbit Fab fragments	Jackson ImmunoResearch	111-007-003	Specificity: IgG (H+L)
Anti- Myeloperoxidase antibody	Dako	A0398
4,6-Diamidino-2-phenylin (DAPI)	Life Technologies Corporation	D1306