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 in the hot water beaker.</li>
<li>Micropipette chemoattractant solution (e.g., 10 &micro;l of 0.5 &micro;g of CXC chemokine MIP-2
or 12 &micro;l of 1 mM WKYMVm) into the lid of a 1.5-mL Eppendorf tube containing 3 &micro;l
India ink and mix well by aspiration using a micropipette (avoid air bubble).</li>
<li>Cut the tip end of a 200-&micro;l pipet tip and micropipette 110 &micro;l of agarose solution (42&deg;C)
into the lid and immediately mix well using another pipette tip (avoid air bubble).</li>
<li>Store the chemoattractant-containing gel at +4&deg;C.</li>
</ol>
2. Preparation of Cremaster Muscle for Intravital Microscopy (Figure 1)
<ol>
<li>Anesthetize an adult male mouse by an i.p. injection of a mixture of 10 mg/kg xylazine
and 200 mg/kg ketamine hydrochloride.</li>
<li>Shave the area over right external jugular vein and the anterior aspect of the scrotum
with an electric razor. After anesthesia, it is important to give special attention and
care to the anesthetized mouse. A heat lamp may be used to prevent the mouse from
hypothermia. The mouse should be free from pain reflex.
</li>
<li>Make a horizontal incision, find and catheterize the jugular vein using a PE-10 tubing
filled with 100 U/mL heparin saline. Catheterization of the jugular vein is needed for the administration of additional anesthetics and drugs when required.</li>
<li>Fix mouse hind legs with umbilical tape with the mouse lying face up on a home-made
cremaster muscle board (Figure 1).
</li>
<li>Connect the board to a 37&deg;C water circulator to keep the cremaster muscle and mouse
body warm.</li>
</ol>

Note: All procedures from 2.6 to 2.14 must be performed very gently5.

<ol start="6">
<li>Make an incision in the scrotal skin to expose the left cremaster muscle. Carefully
dissect the muscle from the associated fascia.
</li>
<li>Superfuse the cremaster muscle with 37&deg;C-warmed bicarbonate-buffered saline (131.9
NaCl, 4.7 KCl, 1.2 MgSO4, 20 NaHCO3, in mM, pH 7.4) using a peristaltic pump.
</li>
<li>Tie a 4&minus;0 suture to the distal end of the cremaster muscle to hold it down on the clear
viewing glass pedestal of the cremaster muscle board.
</li>
<li>Cauterize the cremaster muscle longitudinally. With 4&ndash;0 suture, hold the muscle flat
and secure it along the edges on the pedestal. Separate the testicle and the epididymis
from the underlying muscle and move them into the abdominal cavity.
</li>
<li>Superfuse the muscle at ~0.6 ml/min with the 37&deg;C-warmed perfusion buffer and
cover the exposed muscle with a 22&times;22 mm glass coverslip.
</li>
<li>Place the cremaster muscle board on the microscope stage, examine the muscle
under the microscope, find a suitable postcapillary venule (Select the venule that is straight and unbranched and has
normal shear rate and a diameter in 25&minus;40 &micro;m) and adjust the video camera to allow
the venule to be visualized in a vertical position on either left or right end of the TV
monitor.
</li>
<li>After 30 min's equilibrium, record the video images of the selected postcapillary venule for 5 min as
baseline control data using a video recorder.
</li>
<li>Stop the superfusion and remove the coverslip on the muscle.
</li>
<li>Place an ~1-mm3 sized chemoattractant-containing gel on the surface of the
cremaster muscle in a preselected area 350 &micro;m from and parallel to the observed postcapillary venule,
add coverslip to hold the gel in place and superfuse the muscle tissue at a very slow
rate (&le;10 &micro;l/min) to allow the establishment of a gradient of chemoattractant that is
slowly released and formed from the gel.
</li>
<li>Record the video image for 90 min after the addition of the chemoattractant
containing gel. During the recording, adjust and keep the microscope focus on the
adhering, crawling, transmigrating and chemotaxing leukocytes inside the venule and
in the muscle tissue.
</li>
<li>After the experiment, import the video file to a computer for analysis.
</li>
</ol>

3. Cell Tracking Using ImageJ
<ol><li>On a computer, extract and convert the video to AVI format (e.g., use free computer
software bitRipper to convert DVD video to AVI file).</li>
<li>Use video editing software to generate the time-lapsed movie. For example, use
Windows Movie Maker to make a time-lapsed movie (to 1/512 or 1/1024 time-lapse at 30-fps rate) from the original, real-time
video. Convert and save the uncompressed time-lapsed movie to DV-AVI format.</li>
<li>Record the images of calibration micrometer under the same microscope setting,
import images to the computer, open the images with ImageJ. In ImageJ, the total
pixels appear on the top left of the screen (e.g., 720&times;480 pixels). Measure the size of
the screen at both X and Y axes (e.g., 200&times;150 &micro;m). From this, calculate the number
of pixels per &micro;m (e.g., x = 720/200 = 3.6 pixels/&micro;m, and y = 480/150 = 3.2 pixels/&micro;m).
</li>
<li>To import the movie, open ImageJ again, click &ldquo;File&minus;Import&minus;Using Quicktime Movies
Plug-in&rdquo;, select the movie to be analyzed and click &ldquo;OK&rdquo; at the interface of &ldquo;QT
Movie Opener&rdquo;.
</li>
<li>Click &ldquo;Plugins&minus;Manual Tracking&rdquo; to track cells. Fill in the relevant information into
the fields at the bottom before start tracking. Briefly,
<ol>
<li>Time interval (in sec) = fold-time-lapse/30 (For example, a 1020&times; time-lapse would be 1020/30 = 34 sec/frame interval).
</li>
<li>x/y calibration = the &micro;m/pixel measurement using the calibration of the
image of the micrometer.
</li>
<li>z calibration = 0 (as the mouse cremaster muscle is an extremely thin
layer of tissue and cell crawling and migration are approximately 2D under bright-field transillumination).
</li>
<li>Search square size for centering = 1.
</li>
<li>Dot size/Line width/Font size: they can be adjusted if needed.
</li>
</ol>
</li>
<li>Select a stable and clear point as a reference point. This reference point can be any
clear and small structural point that remains unchanged and stable throughout the
whole experiment. Click &ldquo;Add Track&rdquo; to track the reference point from the first to the
last frame and click &ldquo;End Track&rdquo;. The data appear on results table automatically.

</li>
<li>Track crawling and migrating neutrophils one by one: click &ldquo;Add Track&rdquo; to track the
cell from its appearance in tissue to its disappearance in each frame and click &ldquo;End
Track&rdquo; to finish and save the results in Microsoft Excel.
</li>
</ol>

4. Analysis of Neutrophil Recruitment Parameters

<ol>
<li>Open the results file in Microsoft Excel, and analyze the data (take the changes of
reference point into analysis).
</li>
<li> Intraluminal crawling
<ol>
<li>Crawling distance: the total distance the cell crawls in the lumen from the
initial adherent site to the optimal transmigration site (&micro;m).
</li>
<li>Crawling velocity: crawling distance/time (&micro;m/min).
</li>
</ol>
</li>
<li>Transendothelial migration
<ol>
<li>Transmigration time: from the time the cell starts to transmigrate across
endothelium to the time when the whole cell body is just outside the venule and no cell body can be seen in the lumen (min or sec).
</li>
<li>Detachment time: from the time the whole cell body is just outside the venule
(immediately after transmigration) to the time point when the cell loses contact
with the venule (the tail retracts) (min or sec).
</li>
</ol>
</li>
<li>Chemotaxis in tissue
<ol>
<li>Migration distance: the sum of the distance the cell moves from the start
point to the end point of the migration in tissue (&micro;m).
</li>
<li>Velocity of migration: migration distance in tissue/time (&micro;m/min).
</li>
<li>Chemotaxis distance: the sum of distance the cell migrates in x-axis in
tissue (&micro;m).
</li>
<li>Velocity of chemotaxis: chemotaxis distance/time (&micro;m/min).
</li>
<li>Chemotaxis index: the ratio of dividing the chemotaxis distance by the
migration distance in tissue.
</li>
</ol>
</li>
</ol>
5. Representative Results:
Although bightfield intravital microscopy is used for the study of leukocyte-endothelial cell interactions and may not be necessarily for neutrophils, we confirmed that, by our histology studies, more than 95% of the recruited cells in neutrophil chemoattractant-treated cremaster muscles were indeed neutrophils. In this report, using neutrophil-selective chemoattractants, we present procedures of tracking the recruitment of neutrophils in vivo. Specifically, we describe a protocol of tracking neutrophil intraluminal crawling,
transendothelial migration, and chemotaxis in cremaster muscle tissue in anesthetized
mice using time-lapse intravital video microscopy and ImageJ. The chemoattractant-containing agarose gel on the cremaster muscle slowly releases chemoattractant and allows
a chemoattractant gradient to be established in tissue. Neutrophil chemoattractant induces
neutrophil-endothelial cell interactions in cremasteric postcapillary venules in mice. The
whole experiment is visualized under an upright brightfield intravital microscope with
video images projected by a color video camera to a TV monitor and recorded by a video
recorder. We determined the neutrophil intraluminal crawling, transendothelial migration
and migration and chemotaxis in muscle tissue in response to neutrophil chemoattractant
MIP-2 and WKYMVm prepared in agarose gel (Figure 2). We found that MIP-2 (at
0.5 &micro;M) and WKYMVm (at 0.1 mM) elicited neutrophil intraluminal crawling at similar
velocity, neutrophil transendothelial migration and detachment from the venule for
comparable length of time, neutrophil migration and chemotaxis in muscle tissue at nearly
the same velocity and with similar neutrophil chemotaxis indexes (P > 0.05, Student t test).
<img src="/files/ftp_upload/3296/3296fig1.jpg" alt="Figure 1" /> 
Figure 1. The schematic illustration of an intravital microscope system. The mouse
cremaster muscle is exteriorized on the clear viewing pedestal of cremaster muscle board
on microscope stage and superfused with 37&deg;C-warmed bicarbonate-buffered saline. The
upright microscope is connected with a CCD color video camera for brightfield intravital
microscopy. A monochrome deep-cooled CCD digital camera is also connected to
microscope port for fluorescence intravital microscopy, the images from which are directly
processed by a computer.
<img src="/files/ftp_upload/3296/3296fig2.jpg" alt="Figure 2" /> 
Figure 2. Neutrophil recruitment parameters of brightfield intravital microscopy.
Neutrophil recruitment was induced by the gradual release of neutrophil chemoattractant
MIP-2 or WKYMVm in the agarose gel preparation placed 350 &micro;m adjacent to the
postcapillary venule. Time-lapsed video data were analyzed by ImageJ after processing
the real time video recording of the experiment. Neutrophil intraluminal crawling (A),
transmigration time and detachment time (B), migration velocity and chemotaxis velocity
in tissue (C), and chemotaxis index in cremaster muscle (D) were determined after the
administration of MIP-2 or WKYMVm agarose gel on cremaster muscle in C57BL/6 mice
(n = 3, # of tracked cells = 22 (in A and B) and 27 (in C and D) respectively for MIP-2, and
= 26 (in A and B) and 44 (in C and D) respectively for WKYMVm).

<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>

No conflicts of interest declared.

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.

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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.

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 ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

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 &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; 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.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

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.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

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

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.

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

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.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...