Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Summary

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Abstract

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal — used as a measure of priming 2) in vivo neutrophil labeling — achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Introduction

Neutrophils are the most abundant and short-lived leukocytes in circulation. They are rapidly recruited to the sites of infection or injury, where they serve as professional phagocytes through release of reactive oxygen and nitrogen intermediates along with granules containing antimicrobial peptides and proteases¹. During their recruitment, neutrophils are "primed" by various agents including microbial products, chemoattractants, and inflammatory cytokines, resulting in markedly enhanced phagocyte functionality upon arrival at a site of inflammation². The mechanisms of neutrophil priming have been extensively studied in vitro

Protocol

All animal experiments are performed in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of the University of Toledo.

1. Phenotyping of pIL1-DsRed Mice

NOTE: Offspring are generated by breeding heterozygous pIL1-DsRed mice with wild-type (WT) C57BL6 mice. Three to four week old pups are considered ready for phenotyping. Submandibular bleeding of mice follows an established protocol with minor modifications²³.

1. Isolation of White Blood Cells from the Whole Blood of Mouse Pups
   1. Add 20 µ....

Results

Screening of pIL1-DsRed mice is performed based on the phenotypic DsRed fluorescence signal produced by their peripheral blood leukocytes using flow cytometry. LPS stimulation is known to induce IL-1β production in myeloid cells including neutrophils, monocytes, and dendritic cells^26-28. Thus, isolated leukocytes are incubated with LPS for 4 hr prior to flow cytometry analysis. Next, gating is set for circulating myeloid cells based on cell size (FSC) and internal complexity (S.......

Discussion

The aim of this study is to develop a technology for monitoring the process of neutrophil priming in living animals, which has not yet been fulfilled by the currently available techniques. To achieve this goal, three established methodologies are performed: 1) induction of skin inflammation in IL-1β promoter-driven DsRed reporter mice as a measure of priming, 2) in vivo labeling of neutrophils with low doses of fluorescence-conjugated anti-Ly6G mAb, and 3) intravital confocal microscopy imaging. The combina.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Heparin sodium	APP Pharmaceuticals	NDC 63323-540-31
ACK lysing buffer	Lonza	10-548E
Fetal bovine serum	Sigma-Aldrich	F0926
Lipopolysaccharides	Sigma-Aldrich	L4391
Ketamine hydrochloride	Hospira	NDC 0409-2051-05
Xylazine	LLOYD Laboratory	NADA #139-236
Acepromazine	Boehringer Ingelheim	ANADA 200-361
Hair-removal cream	Church & Dwight
Acetone	Fisher Scientific	A16P4
Oxazolone	Sigma-Aldrich	E0753
Alexa Fluor 647 anti-mouse Ly6G antibody	BioLegend	127610
U-100 insulin syringe with 28 G needle	BD	329461
FITC-CM-Dextran, 150 kDa	Sigma-Aldrich	74817
Butterfly infusion set (27 G needle)	BD	387312
FACSCalibur cytometer	BD
CellQuest Pro software	BD
Confocal microscope	Olympus	FV1000
Metamorph Software	Universal Imaging