This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

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
<ol>
	<li>Sacrifice mouse (8-12 week old) by cervical dislocation. Sterilize hind legs with 70% ethanol. Remove skin from hind legs.</li>
	<li>Dissect out mouse femurs and tibias and remove tissue from legs with a scalpel. Rinse legs wit...

<ol>
	<li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Nourshargh, S., Hordijk, P. L., Sixt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaching+multiple+barriers:+leukocyte+motility+through+venular+walls+and+the+interstitium.">Breaching multiple barriers: leukocyte motility through venular walls and the interstitium.</a> Nat Rev Mol Cell Biol. 11, 366-378 (2010).</li><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+migration+into+inflamed+tissues.">Leukocyte migration into inflamed tissues.</a> Immunity. 41, 694-707 (2014).</li><li>Ley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.+Intravital+microscopic+investigation+of+leukocyte+interactions+with+the+blood+vessel+wall.">Chapter 11. Intravital microscopic investigation of leukocyte interactions with the blood vessel wall.</a> Methods Enzymol. 445, 255-279 (2008).</li><li>Sperandio, M., Pickard, J., Unnikrishnan, S., Acton, S. T., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+leukocyte+rolling+in+vivo+and+in+vitro.">Analysis of leukocyte rolling in vivo and in vitro.</a> Methods Enzymol. 416, 346-371 (2006).</li><li>Auffray, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+blood+vessels+and+tissues+by+a+population+of+monocytes+with+patrolling+behavior.">Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior.</a> Science. 317, 666-670 (2007).</li><li>Cros, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD14dim+monocytes+patrol+and+sense+nucleic+acids+and+viruses+via+TLR7+and+TLR8+receptors.">Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors.</a> Immunity. 33, 375-386 (2010).</li><li>Hanna, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+NR4A1+(Nur77)+controls+bone+marrow+differentiation+and+the+survival+of+Ly6C-+monocytes.">The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes.</a> Nat Immunol. 12, 778-785 (2011).</li><li>Woodfin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+junctional+adhesion+molecule+JAM-C+regulates+polarized+transendothelial+migration+of+neutrophils+in+vivo.">The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.</a> Nat Immunol. 12, 761-769 (2011).</li><li>Proebstl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+support+neutrophil+subendothelial+cell+crawling+and+breaching+of+venular+walls+in+vivo.">Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo.</a> J Exp Med. 209, 1219-1234 (2012).</li><li>Wantha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-derived+cathelicidin+promotes+adhesion+of+classical+monocytes.">Neutrophil-derived cathelicidin promotes adhesion of classical monocytes.</a> Circ Res. 112, 792-801 (2013).</li><li>Ozinsky, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repertoire+for+pattern+recognition+of+pathogens+by+the+innate+immune+system+is+defined+by+cooperation+between+toll-like+receptors.">The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.</a> Proc Natl Acad Sci U.S.A. 97, 13766-13771 (2000).</li><li>Lammermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+swarms+require+LTB4+and+integrins+at+sites+of+cell+death+in+vivo.">Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo.</a> Nature. 498, 371-375 (2013).</li><li>Carlin, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nr4a1-dependent+Ly6C(low)+monocytes+monitor+endothelial+cells+and+orchestrate+their+disposal.">Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal.</a> Cell. 153, 362-375 (2013).</li><li>Hamon, P., Rodero, M. P., Combadiere, C., Boissonnas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+mouse+bone+marrow+monocytes+in+vivo.">Tracking mouse bone marrow monocytes in vivo.</a> J Vis Exp. , (2015).</li><li>Iqbal, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD68+promoter+GFP+transgenic+mice+allow+analysis+of+monocyte+to+macrophage+differentiation+in+vivo.">Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.</a> Blood. 124, e33-e44 (2014).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PloS One. 6, e25109 (2011).</li></ol>

The authors declare that they have no competing financial interests.

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...

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 implicated in the leukocyte recruitment cascade, it is important to visualize the kinetics of cell recruitment and to track the behavior of each cell/population. An effective method involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
To date, several approaches using intravital microscopy were developed to image the vasculature 4,5. For instance, imaging of ear dermis vasculature or mesenteric veins by intravital confocal microscopy was used to identify the patrolling behavior of murine Ly6Clow monocytes and human CD14dimCD16+ monocytes on the luminal side of blood vessels under steady state conditions 6-8. The mouse cremaster vasculature model is often used to monitor the behavior of neutrophils or inflammatory Ly6Chigh monocytes under inflammatory or ischemic conditions in transgenic mice. In that case, cremaster is stimulated via intrascrotal injection of IL1&#946;, CCL2, TNF&#946; or fMLP. After 2-4 hr, tissues are then surgically exteriorized and analyzed by intravital confocal microscopy 9-11.
The following protocol describes a method to monitor monocytes and neutrophils at the same time with any inverted fluorescence confocal microscope. Furthermore, our method details how to image the same vessel before (steady state condition) and after inflammation and how to follow the kinetics of leukocyte recruitment. To this purpose, we use the CX3CR1gfp/wt mouse, whose monocytes express eGFP, i.v. injected with an orange dye-labeled murine neutrophils. Using an inverted confocal microscope, it is possible (1) to track and analyze the patrolling Ly6Clow monocytes under steady state conditions and (2) to follow the recruitment of both monocytes and neutrophils in the same vessel after local inflammation. Here we create the inflammatory conditions by using the TLR2/TLR1 agonist Pam3CSK4 12. Moreover, such imaging may help to determine the role of specific molecules of interest in the various steps of the leukocyte recruitment cascade if performed on specific knock-out mice or in presence of blocking antibodies 6,9,13.

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

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 CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;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.

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 &#176;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 ar...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K ビュー.  University of Geneva. 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.