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 with 90% ethanol and place into a culture dish filled with PBS.</li>
	<li>Under the culture hood, clean tissue from femurs and tibias with a scalpel and separate at knee joint. Cut off each end of the bones.</li>
	<li>Flush bone marrow from each bone using a 23G needle and a 1 ml syringe filled with preparation medium (phenol red-free DMEM/F12, 1% rat serum), into a 50 ml screw tube. Disrupt cell aggregates by delicately passaging through the 23G needle and a 1 ml syringe. Bring total volume to 25 ml with preparation medium.</li>
	<li>Centrifuge 250 x g, 5 min, 4 &#176;C. Discard supernatant. Resuspend pellet in 1 ml Red Blood Cell Lysis RBLC buffer (8.3 g NH4Cl, 1 g NaHCO3, 1 ml EDTA (100 mM), complete to 1 L with distilled water, filter 0.22 &#181;m) in a 15 ml screw tube. Incubate 30 sec&#160;on ice.</li>
	<li>Add 10 ml of preparation medium and centrifuge 250 x g, 5 min, 4 &#176;C. Discard supernatant and resuspend in 2 ml preparation medium.</li>
</ol>
2. Neutrophil Enrichment by Negative Selection
<ol>
	<li>Add 150 &#181;l of Mouse neutrophil enrichment cocktail (cell isolation platform kit such as EasySep). Mix and incubate on ice for 10 min.</li>
	<li>Add 10 ml of phenol red free DMEM. Invert once and centrifuge 250 x g, 3 min, 4 &#176;C.</li>
	<li>Discard supernatant. Resuspend pellet in 1.75 ml preparation medium in 5 ml polystyrene round bottom tubes. Add 250 &#181;l Biotin Selection Cocktail. Mix and incubate on ice for 10 min.</li>
	<li>Vortex the magnetic particles (from the cell isolation platform kit) for 30 sec to resuspend particles. Add 500 &#181;l magnetic particles to cell suspension. Mix and incubate on ice for 10 min.</li>
	<li>Place the tube into the magnet. Wait 3 min.</li>
	<li>Invert the magnet onto a new 5 ml polystyrene round bottom tubes to collect the desired unlabeled cells. Do not take the last drop hanging in the tube. Unwanted cells will remain in the tube inside the magnet. Discard this tube into a biohazard waste.</li>
	<li>Place the new tube into the magnet. Wait 3 min.</li>
	<li>Invert the magnet onto a new 15 ml screw tube to collect the desired unlabeled cells. Do not take the last drop hanging in the tube.</li>
	<li>Complete volume to 5 ml with phenol red free DMEM.</li>
</ol>
3. Labeling of Neutrophils
<ol>
	<li>Add 0.5 &#181;l of an orange dye such as Cell Tracker Orange (working concentration 1 &#181;M, CMRA &#8211; chloromethyl-rodol-acetate, stock 10 mM in DMSO). Incubate 2 min at 37 &#176;C.</li>
	<li>Add 2.5 ml of preparation medium. Centrifuge 250 x g, 5 min, 4 &#176;C.</li>
	<li>Discard supernatant. Resuspend pellet in 1 ml preparation medium in a 1.5 ml tube.</li>
	<li>Take an aliquot to count with hematocytometer.</li>
	<li>Incubate 5 min at 37 &#176;C. Centrifuge 250 x g, 5 min, 4 &#176;C.</li>
	<li>Discard supernatant. Resuspend pellet in 1 ml PBS. Centrifuge 250 x g, 5 min, 4 &#176;C to wash.</li>
	<li>Discard supernatant. Resuspend pellet as 10x106 cells per 100 &#181;l PBS. Keep on ice until i.v. injection. NOTE: cells have to be i.v. injected as quickly as possible. 6-12x106 neutrophils/mouse are usually obtained.</li>
</ol>
4. Mouse Preparation for Intravital Microscopy
NOTE: Step 4.1.) and 4.2.) should be performed at the beginning of the experiment to avoid extended waiting time of labeled neutrophils on ice.
<ol>
	<li>Weigh the CX3CR1gfp mouse (8-12 week old).</li>
	<li>Prepare 800 &#181;l of anesthetics (ketamine 60 mg/kg, xylazine 4.5 mg/kg, acepromazone 1.75 mg/kg in PBS).</li>
	<li>Anesthetize mouse. Injection i.p. with 200 &#181;l / 20 g of mouse. Control anesthesia by toe pinching and by checking the respiratory rate.</li>
	<li>Inject neutrophils (10x106 cells in 100 &#181;l PBS) intravenously using the lateral tail veins or the retro-orbital sinus, at the convenience of the experimenter.</li>
	<li>Put the mouse on heating pad and protect the eyes with ocular gel.</li>
	<li>A glass coverslip (35 mm diameter) is put in a 10 cm tissue culture dish, which has a 30 mm diameter hole. Use oil is to maintain the tissue culture dish in contact with the coverslip.</li>
	<li>Incise abdominal skin with scissors to expose the peritoneum. Incise peritoneum with scissors to expose intestine.</li>
	<li>Add 200 &#181;l PBS (pre-heated at 37 &#176;C) into the peritoneal cavity. Take the mouse in your hand and turn it face down, so that the intestine gets out of the peritoneal cavity with a gentle pressure. Place the mouse in the tissue culture dish.</li>
	<li>Gently deroll the intestine with sterilized cotton buds onto the coverslip in order to expose the mesenteric vessels.</li>
	<li>Cut paper tissues in bands and wet them with 37&#176;C heated PBS. Immobilize the intestine with the pieces of paper tissues to decrease the movements resulting from peristalsis.</li>
	<li>Put the tissue culture dish and the mouse inside the 37 &#176;C thermostated chamber onto the custom-made tray stage of the inverted microscope. Introduce a syringe containing anesthetics s.c. into the hind leg. NOTE: Additionally, the mouse can receive oxygen (0.5 L/min) with a mask.</li>
	<li>Control mouse for anesthesia by toe pinching and checking the respiratory rate every 30 min. If needed, deliver a boost of anesthetics (20 &#181;l) to properly maintain anesthesia. NOTE: Drying of the vasculature should be avoided. Therefore, its humidity is maintained by regularly wetting the paper tissues used to immobilize the intestine with 37&#176;C-warmed PBS.</li>
</ol>
5. Measuring Neutrophil and Monocyte Behavior in Inflamed Vessels In Vivo using Intravital Microscopy
<ol>
	<li>Set lasers to 488- and 561- at the lowest power necessary to avoid laser-induced phototoxicity. In our experiments, it is lower than 0.5%. Intensity of GFP in monocytes and orange dye in neutrophils is so bright that usually 0.3% is enough. NOTE: This represents a laser power of 0.15 to 0.25 mW with our system.</li>
	<li>Acquire images of 512 x 512 pixels (i.e., 635 &#181;m) in 2.2 sec&#160;with an open pinhole using the 20X dry objective of the inverted microscope. Use a z-depth between 10 to 20 &#956;m, that ensures sufficient signal with the low laser powers. 
	NOTE: We usually perform experiments with a z-depth between 12 to 16 &#956;m. In the case of presented figures and movies, the z-depth is 20 &#956;m. Phase contrast allows the identification of endothelial walls and imaging is performed on mesenteric veins.&#160;Since patrolling cells move quite slowly with a speed of 5-10 &#181;m /min 6, recording a field of interest every 20 sec&#160;is satisfactory. With the described settings, the motorized stage allows the recording of up to 7 fields of interest at the same time.</li>
	<li>Place the mouse and the mesenteric vessels in the thermostat chamber of the microscope are allow them to recover from the preparation for 30 min&#160;before the acquisition of the first movie. During this time, choose several fields of interest. Focus on the luminal surface of the vessel closest to the objective. If needed, take advantage of the slight auto-fluorescence of endothelium to set the focus. 
	NOTE: Surgery during mouse preparation slightly stresses the endothelium. Consequently, rolling of neutrophils and/or monocytes can be observed in the first 15 min&#160;following mouse preparation.</li>
	<li>Record a first movie for 30 min&#160;under steady state conditions, one image every 20 sec. 
	NOTE: The software acquires each field of interest in 2.2 sec&#160;and the motorized stage automatically moves from one field to the other. It goes back to the first position within 20 sec&#160;during the 30 min&#160;of the recording movie.</li>
	<li>To initiate blood vessel inflammation, add a drop of 20 &#181;l of PBS containing 100 &#181;g of TLR2/TLR1 agonist Pam3CSK4 12 directly onto the imaged vessels.</li>
	<li>Control the focus for each field of interest. 
	NOTE: During the course of acquisition, there is always slight changes of focus on z-axis. Despite the 10-20 &#956;m z-depth, this can result in a decrease of fluorescence intensity. Therefore, manually refocus each field of interest at the end of each movie, if needed. Record movies for 30 min-periods and up to 3 hr total after initiation of inflammation.</li>
	<li>At the end of the experiment, sacrifice anesthetized mouse by cervical dislocation.</li>
</ol>
6. Generation of Movie Files and Tracking of Leukocytes
NOTE: The Nikon acquisition software generates .nd2 files but the following procedure would be similar with other software and brands.
<ol>
	<li>Export files as tiff series.</li>
	<li>Go to ImageJ software (<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a>). Import the tiff series. Each channel as a different file.</li>
	<li>Apply a median filter with a radius of 2.0 pixel for the green and red channels to reduce background noise (Process &#62; Filters &#62; Median&#8230;).</li>
	<li>Convert files into binary (Process &#62; Binary &#62; Make Binary). Enable the software to calculate the threshold for each image.</li>
	<li>Merge channels into one image series (Image &#62; Color &#62; Merge Channels&#8230;). Select the monocytes in the green channel, the neutrophils in the red channel and the transmitted light in the grey channel.</li>
	<li>Convert stacked images into RGB colors (Image &#62; Color &#62; Stack to RGB).</li>
	<li>Save file as .avi</li>
	<li>Data are imported and compiled in Imaris software (Bitplane). Movements of monocytes and neutrophils are then tracked using the Spot Tracking Wizard.</li>
</ol>

<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. 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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 mesenteric veins.
Our protocol involves the exteriorization of mesenteric vessels before the start of the experiments, enabling (1) to directly induce inflammation on the monitored vessels, and (2) to record the early time points after stimulation and (3) to follow up the kinetics of recruitment of leukocytes and their behavior in the same vessel before and after induction of inflammation.
We transfer neutrophils purified from murine bone marrow of the same genetic background and not from blood as more cells can be obtained per animal (6-12x106 vs 0.5-2x106 /mouse). If use of blood neutrophils is preferred, the same protocol can be carried out for cell purification.
In intravital imaging, the visualization of the vasculature is enabled by the transmitted light in the case of mesenteric vessels. Alternatively, fluorescent antibodies against PECAM1 (clone 390) or fluorescent high molecular weight dextran (70kDa or 2 MDa) 6,14 can be i.v. injected to detect the vasculature. For instance, intra-scrotal injection of 3 &#956;g anti-PECAM1 (clone 390) was reported to enable vasculature detection with low background and not to affect leukocyte activity in the cremaster model 9. Compared to transmitted light, the use of fluorescent antibodies or dyes notably introduces the possibility of 3D reconstruction of the vasculature, but a channel is sacrificed to its labeling and the acquisition time is increased. Transmitted light is sufficient in the mesenter model thanks to the transparency of the vasculature, as described here. On the contrary, fluorescent labelling of the vasculature is mandatory in the ear dermis or opaque organs.
Detection of populations of leukocytes can be achieved using transgenic animals which exhibit endogenous fluorescence in specific cell types (monocytes Cx3cr1eGFP/wt6 or cd115CFP15 or cd68gfp16 ; monocytes and neutrophils LyseGFP9 ; and platelets cd41YFP17). Although CX3CR1 is expressed in all monocytes and some subsets of NK and T cells, the Cx3cr1eGFP/wt mouse is interesting to distinguish between inflammatory Ly6Chigh monocytes which exhibit less GFP fluorescence than resident Ly6Clow monocytes 6. As CD115 is specific of the monocytic line, the MacBlue mouse cd115CFP is an interesting model. However it is not possible to distinguish Ly6Chigh and Ly6Clow monocytes.
An alternative to transgenic fluorescent mice is the i.v. injection of fluorescent antibodies to label the populations of leukocytes in real time. For instance, monocytes can be labeled with anti-CD115 (clone AFS98), neutrophils with anti-Ly6G (clone 1A8) and platelets with anti-CD49b (clone HM&#945;2). In general, all these antibodies are depleting at high dose. Therefore, they should be used with caution for short period of times and at low levels. It should be noted that even at low dose, an impact on cell function cannot be ruled out. Personal experience with low amounts (1-2 &#956;g) enables the labelling of CD115+ monocytes and Ly6G+ neutrophils without depletion (data not published).
Although the model of the ear dermis vasculature is non-invasive as the ear is untouched, the depth of the ear dermis is a concern, which makes it a model more suitable for 2-photon microscopy 13. Moreover, it is not possible to directly stimulate the vessel in the ear model without disturbing physically the preparation.
Our protocol can be applied with any inverted confocal microscope. Since scanning speed and laser power are machine-dependent, acquisition of one frame (512 x 512 pixels, i.e., 635 &#181;m) at a single z-plane requires approximately 2.2 sec&#160;with our settings for the galvano scanner of the Nikon A1R. Unfortunately, this low speed limits the possibility of recording four-dimensional movies of mesenteric veins without a loss of quality. The resonant scanner of the A1R offers a high acquisition speed, although the quality of images is strongly reduced (background versus specific signal) for our experiments with the CX3CR1gfp/wt mouse. It should be kept in mind that lasers are set at lower than 0.5% (&#60; 0.25 mW) to avoid phototoxicity and bleaching.
Woodfin et al managed to acquire movies in 4D (60 z-stack images in 40 sec) of cremaster venules using a Leica TCS-SP5 9. As the authors image values of 20&#8211;40 &#181;m in diameter, they are probably less sensitive to changes in focus than in our model (monitoring of mesenteric veins of 200-400 &#181;m). When imaging live tissue, control of focus is the most important concern, which restricts the possibility to record a unique movie lasting several hours. We consider that recording movies lasting 30 min&#160;(or 45 min) is the most practical in the mesenteric model.
The addition of TLR agonists alters the vessel width and therefore the focus. Consequently, it is very difficult to image properly the first 10-15 min&#160;after stimulation until the vessels stabilize.
Another limitation is the duration of the experiment. Because of the difficulty to maintain the mouse anesthetized properly over a long period of time, it is difficult to image more than 4 hr after inflammation. The use of other anesthetics, such as continuous isoflurane inhalation, might be an option (although not tested by us).
In old mice (over 16 weeks of age), increased amount of fat deposits around the vessels decrease the quality of visualization of mesenteric vessels. Therefore, it is better to run experiments with 8-12 week-old mice.
This technique can be combined with the i.v. injection of antibodies to block molecules of interest or deplete given leukocyte populations, so that the importance of molecules implicated in leukocyte recruitment or behavior can be determined. Additionally, other leukocyte subsets can be tracked using other transgenic mouse strains expressing fluorescent proteins in cell populations of interest.

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 arrow) and arteries (blue arrow).
Treatment of acquired images with ImageJ software generated movie files, such as Movie 1 and Movie 2. Figure 3 displays the different steps of image processing for the first time point of Movie 1. Movie 1 shows the patrolling of Ly6Clow monocytes under steady state conditions, as previously described 6,8,14 whereas all neutrophils are circulating in the bloodstream. No neutrophil is patrolling the endothelial wall. Movie 2 shows the same field of interest 90 min&#160;after the addition of the TLR2/TLR1 agonist Pam3CSK4 to locally initiate inflammation. In this case, neutrophils and monocytes are massively recruited to the endothelial wall, which they scan meticulously. It is important to note that addition of a TLR agonist induces changes in endothelium width and consequently alters the focus of the z-axis. Therefore, focus should be carefully controlled after the addition of the agonist. Compilation of data in Imaris software (Bitplane) allows the tracking of individual leukocytes using the Spot Tracking Wizard. Figure 4 shows the exact track paths of monocytes (green) and neutrophils (red) patrolling along the endothelium. The data are obtained from the analysis of Movie 2, i.e., 90 min&#160;after the initiation of inflammation. Tracking of leukocyte paths can be exported as tiff files or xls files to get various statistics such as track length, displacement, duration or speed.
This technique offers a nice way to monitor the behavior of monocytes and neutrophils at the same time before and after inflammation. It is possible to track the recruitment of these cells on the endothelial wall overtime. Loss of focus on the z-axis or even x/y displacement can occur as a consequence of intestinal movements that ruin the experiment. Movie 3 illustrates such failed preparation and acquisition.
<img alt="Figure 1" src="/files/ftp_upload/53314/53314fig1.jpg" /> 
Figure 1.&#160;Mouse preparation. 
Black arrows indicate PBS-wetted tissue used to immobilize the intestine. White arrowhead indicates the 10cm tissue culture dish. T indicates the aluminum custom-made tray stage that fits into the microscope. Mesenteric blood vessels are nicely exposed at the center on the coverslip. <a href="https://www.jove.com/files/ftp_upload/53314/53314fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53314/53314fig2.jpg" /> 
Figure 2.&#160;Branches of the mesenteric veins and arteries. 
Images (20 x 10) taken with a 10X objective were stitched to constitute one large image of the available area. Several fields of interest are then chosen to monitor leukocyte movement with a 20X objective. The red arrow indicates a mesenteric vein. The blue arrow indicates a mesenteric artery. The green arrow indicates the fat tissue surrounding the vessels. The dark area on the sides of the image results from the presence of wetted tissue used to immobilize the intestine. White scale bar represents 1 mm. <a href="https://www.jove.com/files/ftp_upload/53314/53314fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53314/53314fig3.jpg" /> 
Figure 3.&#160;Image processing with ImageJ. 
The different steps of image processing are illustrated for the first image of Movie 1. Green (monocytes) and red (neutrophils) channels are processed separately. To the original raw data (A), a median filter is applied (B) and then converted to a binary image (C). (D) Channels are merged into a final image. Green or red lines observed in raw data are cells that are moving so fast that the microscope is not able to wholly acquire them. White scale bar represents 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53314/53314fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53314/53314fig4.jpg" /> 
Figure 4.&#160;Individual leukocyte tracks. 
Tracks of monocytes (A) and neutrophils (B) patrolling the endothelium are processed with Imaris Software (Bitplane). White scale bar represents 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53314/53314fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/53314/53314mov1.jpg" /> 
Movie 1.&#160;Leukocytes scanning the endothelial wall at the steady state. At the steady state, Ly6Clow monocytes patrol the endothelium. Neutrophils are circulating according to the blood flow. Neutrophils are in red and monocytes are in green. Scale bar represents 50 &#181;m. Generated with the 20X objective. <a href="https://www.jove.com/files/ftp_upload/53314/Movie1.avi" target="_blank">Please click here to view this video.</a>
<img alt="Movie 2" src="/files/ftp_upload/53314/53314mov2.jpg" /> 
2: Leukocytes scanning the endothelial wall after TLR2-mediated inflammation. Same field of interest as in Movie 1. Movie starts 90 min&#160;after the addition of 20 &#181;l of PBS containing 100 &#181;g of Pam3CSK4 (TLR2/TLR1 agonist) directly onto the vessel. Recruited monocytes and neutrophils are meticulously scanning the endothelial wall. Neutrophils are in red and monocytes are in green. Scale bar represents 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53314/Movie2.avi" target="_blank">Please click here to view this video.</a>
<img alt="Movie 3" src="/files/ftp_upload/53314/53314mov3.jpg" /> 
Movie 3: Results obtained from incorrect mouse preparation. Movements of vessels obtained when anesthesia or immobilization of intestine are incorrect. Scale bar represents 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/53314/Movie3.avi" target="_blank">Please click here to view this video.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.4K Views.  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.

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

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.

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 ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

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. ...