This work was supported by a research grant from Canadian Institutes of Health
Research (CIHR, MOP-86749). L. Liu is a recipient of CIHR New Investigator Award
(MSH-95374).

1. Preparation of Chemoattractant in Agarose Gel
<ol>
<li>Pipette 10 mL of 2&times;PBS in a 50-mL conical tube, and warm up the tube by putting it into
a beaker containing hot water.</li>
<li>Pipette 10 mL of distilled water and add 0.4 g agarose powder in another 50-mL conical
tube (with its cap slightly loosened) and heat the mixture until just boiling in a
microwave oven (for ~1 min in a 700-watt microwave oven).</li>
<li>Add the warmed 2&times;PBS to agarose solution tube, swirl the mixed solution and keep it
warm ...

<ol>
	<li>Liu, L., 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=Molecular+mechanisms+of+leukocyte+recruitment:+organ-specific+mechanisms+of+action.">Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action.</a> Thromb. Haemost. 89, 213-220 (2003).</li><li>Petri, B., Phillipson, M., 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+physiology+of+leukocyte+recruitment:+an+in+vivo+perspective.">The physiology of leukocyte recruitment: an in vivo perspective.</a> J. Immunol. 180, 6439-6446 (2008).</li><li>Phillipson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+crawling+of+neutrophils+to+emigration+sites:+a+molecularly+distinct+process+from+adhesion+in+the+recruitment+cascade.">Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.</a> J. Exp. Med. 203, 2569-2575 (2006).</li><li>Wong, C. H., Heit, 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=Molecular+regulators+of+leucocyte+chemotaxis+during+inflammation.">Molecular regulators of leucocyte chemotaxis during inflammation.</a> Cardiovasc. Res. 86, 183-191 (2010).</li><li>Gavins, F. N., Chatterjee, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+for+the+study+of+mouse+microcirculation+in+anti-inflammatory+drug+research:+focus+on+the+mesentery+and+cremaster+preparations.">Intravital microscopy for the study of mouse microcirculation in anti-inflammatory drug research: focus on the mesentery and cremaster preparations.</a> J. Pharmacol. Toxicol. Methods. 49, 1-14 (2004).</li><li>Bullen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+imaging+techniques+for+drug+discovery.">Microscopic imaging techniques for drug discovery.</a> Nat. Rev. Drug Discov. 7, 54-67 (2008).</li><li>Hazelwood, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entering+the+Portal:+Understanding+the+Digital+Image+Recorded+Through+a+Microscope.">Entering the Portal: Understanding the Digital Image Recorded Through a Microscope.</a> Imaging Cellular and Molecular Biological Functions. , 3-43 (2007).</li><li>Hickey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-selectin+facilitates+emigration+and+extravascular+locomotion+of+leukocytes+during+acute+inflammatory+responses+in+vivo.">L-selectin facilitates emigration and extravascular locomotion of leukocytes during acute inflammatory responses in vivo.</a> J. Immunol. 165, 7164-7170 (2000).</li><li>Cara, D. C., 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=Intravital+microscopy+as+a+tool+for+studying+recruitment+and+chemotaxis.">Intravital microscopy as a tool for studying recruitment and chemotaxis.</a> Methods Mol. Biol. 239, 123-132 (2004).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSP1+is+an+endothelial+gatekeeper+of+leukocyte+transendothelial+migration.">LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration.</a> J. Exp. Med. 201, 409-418 (2005).</li><li>Heit, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+accelerates,+but+is+not+required+for,+neutrophil+chemotaxis+to+fMLP.">PI3K accelerates, but is not required for, neutrophil chemotaxis to fMLP.</a> J. Cell Sci. 121, 205-214 (2008).</li></ol>

Intravital microscopy is the essential tool for revealing the cellular and molecular
mechanisms of leukocyte recruitment during inflammation. Quantitative visualization
for determination of leukocyte-endothelial cell interactions in microvasculature of
transluscent tissues such as cremaster muscle and mesentery remains the gold standard for
the application of the technique1,5. The conventional brightfield intravital microscopy has
many unique technical features and the recruitment mechanisms revealed in these ...

Table: Materials, Specific Reagents and Equipment
<table border="1">
	<tr>
		<td>
			Name of the reagent/equipment
		</td>
		<td>
			Company
		</td>
		<td>
			Catalogue number
		</td>
		<td>
			Comments
		</td>
	</tr>
	<tr>
		<td>
			Polyethylene
			tubing, PE10
		</td>
		<td>
			Becton
			Dickinson
		</td>
		<td>
			427401
		</td>
		<td>
			I.D.
			0.28mm &times;
			O.D. 0.61mm
		</td>
	</tr>
	<tr>
		<td>
			India
			ink
		</td>
		<td>
			Speedball
			Art
		</td>
		<td>
			Super
			Black
		</td>
		<td>
			100%
			carbon black pigment-no
			dyes
		</td>
	</tr>
	<tr>
		<td>
			Cautery
		</td>
		<td>
			Aaron
			Medical
		</td>
		<td>
			AA03
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Xylazine
		</td>
		<td>
			Bayer
			HealthCare, Bayer Inc.
		</td>
		<td>
			DIN
			02169592
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Ketamine
			hydrochloride
		</td>
		<td>
			Bioniche
			Animal Health Canada, Inc.
		</td>
		<td>
			DIN
			01989529
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Murine
			recombinant MIP-2
		</td>
		<td>
			R&amp;D
			Systems
		</td>
		<td>
			452-M2
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			WKYMVm
		</td>
		<td>
			Phoenix
			Pharmaceuticals, Inc.
		</td>
		<td>
			072-12
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Agarose
		</td>
		<td>
			Invitrogen
		</td>
		<td>
			15510-027
		</td>
		<td>
			Ultrapure
		</td>
	</tr>
	<tr>
		<td>
			Heparin
		</td>
		<td>
			Sigma
		</td>
		<td>
			H-3393
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Upright
			microscope
		</td>
		<td>
			Olympus
		</td>
		<td>
			BX61WI
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			3CCD
			color
			video camera
		</td>
		<td>
			SONY
		</td>
		<td>
			DXC-990
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			HD-DVD
			video recorder
		</td>
		<td>
			LG
			Electronics Inc.
		</td>
		<td>
			RH398H-M
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			TV
			monitor
		</td>
		<td>
			LG
			Electronics Inc.
		</td>
		<td>
			22LG30
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Water
			circulator
		</td>
		<td>
			Thermo
			Scientific
		</td>
		<td>
			HAAKE
			DC10
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Peristaltic
			pump
		</td>
		<td>
			Gilson;
			Pharmacia
		</td>
		<td>
			Gilson
			MINIPULS 3;
			Pharmacia
			P-3
		</td>
		<td>&nbsp;</td>
	</tr>
	<tr>
		<td>
			Cremaster muscle board
		</td>
		<td>
			University
			of Saskatchewan
		</td>
		<td>&nbsp;</td>
		<td>
			Home-made
		</td>
	</tr>
</table>

tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

Watch this Scientific Journal Video about Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy at JoVE.com

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

tracking-neutrophil-intraluminal-crawling-transendothelial-migration

Research

JoVE Journal

Immunology and Infection

19.5K Views.  University of Saskatchewan. We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.