Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Summary

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Abstract

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.

Here we present a protocol for imaging monocytes and neutrophils in the CX₃CR1^gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.

The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

Introduction

Neutrophils and monocytes are cells of the innate immune system that continuously circulate in the blood. Upon injury or infection, inflammatory signals induce leucocyte diapedesis into damaged and infected tissues, herein initiating a cellular immune response ^1-3. The rapidity of leukocyte mobilization determines the positive outcome of the immune responses. These intricate processes rely on specific molecules (e.g., selectins, endothelium-bound chemokines) present on the inflamed endothelium that help for the establishment of adhesive contacts between circulating leukocytes and the endothelium ^1-3.To get insights on the molecules impli....

Protocol

NOTE: Animal procedures were performed in accordance with the Institutional Ethical Committee of Animal Care in Geneva, Switzerland and the Cantonal Veterinary Office. Authorization number GE/63/14.

1. Preparation of a Single Cell Suspension from Bone Marrow

1. Sacrifice mouse (8-12 week old) by cervical dislocation. Sterilize hind legs with 70% ethanol. Remove skin from hind legs.
2. Dissect out mouse femurs and tibias and remove tissue from legs with a scalpel. Rinse legs with 90% ethanol and place into a culture dish filled with PBS.
3. Under the culture hood, clean tissue from femurs and tibias with a scalpel and ....

Results

The manuscript describes an optimized protocol to easily monitor the behavior of monocytes and neutrophils in the mesenteric veins of anaesthetized mice in real time. The use of a 37 °C-thermostated chamber is mandatory to maintain the temperature of the mouse and also due to the temperature dependent movement of leukocytes. Preparation of the mouse is displayed in Figure 1. Figure 2 shows all the area seen under the microscope. Transmitted light allows the identification of mesenteric veins (red arrow) .......

Discussion

The methodologies described in this manuscript provide a consistent approach to efficiently study monocyte and neutrophil behavior in mesenteric veins under steady state and inflammatory conditions.

The crucial step of the preparation is the immobilization of the intestine with PBS-wetted tissues. If performed properly, mesenteric vessels are nicely exposed on the coverslip for image acquisition. This enables the selection of several fields of interest to monitor leukocyte behavior in mesenter.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5 ml polystyrene round bottom tubes	Beckton Dickinson	352058
10x CFI Plan Apochromat 0.45   DT:4mm	Nikon
20x CFI Plan Apochromat VC 0.75 DT:1mm	Nikon
Cell Tracker Orange CMRA Dye	Life Technologies	C34551
EasySep Magnet	Stem Cell Technologies	18000
EasySep Mouse Neutrophil Enrichement kit	Stem Cell Technologies	19762
EDTA	Sigma Aldrich	E6758
FCS	PAA	A15-042
Immersion Oil Type A	Nikon		any viscous oil
Life Box Temperature Control System	Life Imaging
NaHCO3	Sigma Aldrich	S5761
NH4Cl	Sigma Aldrich	A9434
Nikon A1R confocal microscope	Nikon	A1R	inverted microscope, motorized x/y/z stage, NIS elements software
PBS	Life Technologies	D8537
phenol red free DMEM/F12	Life Technologies	21041-025	any phenol red free medium is suitable
PAM3CSK4	Invivogen	tlrl-pms	reconstitute in PBS
Rat serum	Stem Cell Technologies		included in EasySep Mouse Neutrophil Enrichement kit
Tissue culture dish 100	TPP	93100