Name: Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time (Video) | JoVE Journal
Uploaded: 2015-11-16T13:00:00.000+00:00
Duration: 9 min 28 s
Description: Learn intravital confocal microscopy to monitor neutrophil and monocyte dynamics in mouse mesenteric veins under steady state and inflammatory conditions. This method enables real-time tracking of leukocyte migration and recruitment.

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5 minutes

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>

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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 mesenter...

Neutrophils and monocytes are cells of the innate immune system that continuously circulate in the blood. Upon injury or infection, inflammatory signals induce leucocyte diapedesis into damaged and infected tissues, herein initiating a cellular immune response 1-3. The rapidity of leukocyte mobilization determines the positive outcome of the immune responses. These intricate processes rely on specific molecules (e.g., selectins, endothelium-bound chemokines) present on the inflamed endothelium that help for the establishment of adhesive contacts between circulating leukocytes and the endothelium 1-3.To get insights on the molecules implicated in the leukocyte recruitment cascade, it is important to visualize the kinetics of cell recruitment and to track the behavior of each cell/population. An effective method involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
To date, several approaches using intravital microscopy were developed to image the vasculature 4,5. For instance, imaging of ear dermis vasculature or mesenteric veins by intravital confocal microscopy was used to identify the patrolling behavior of murine Ly6Clow monocytes and human CD14dimCD16+ monocytes on the luminal side of blood vessels under steady state conditions 6-8. The mouse cremaster vasculature model is often used to monitor the behavior of neutrophils or inflammatory Ly6Chigh monocytes under inflammatory or ischemic conditions in transgenic mice. In that case, cremaster is stimulated via intrascrotal injection of IL1&#946;, CCL2, TNF&#946; or fMLP. After 2-4 hr, tissues are then surgically exteriorized and analyzed by intravital confocal microscopy 9-11.
The following protocol describes a method to monitor monocytes and neutrophils at the same time with any inverted fluorescence confocal microscope. Furthermore, our method details how to image the same vessel before (steady state condition) and after inflammation and how to follow the kinetics of leukocyte recruitment. To this purpose, we use the CX3CR1gfp/wt mouse, whose monocytes express eGFP, i.v. injected with an orange dye-labeled murine neutrophils. Using an inverted confocal microscope, it is possible (1) to track and analyze the patrolling Ly6Clow monocytes under steady state conditions and (2) to follow the recruitment of both monocytes and neutrophils in the same vessel after local inflammation. Here we create the inflammatory conditions by using the TLR2/TLR1 agonist Pam3CSK4 12. Moreover, such imaging may help to determine the role of specific molecules of interest in the various steps of the leukocyte recruitment cascade if performed on specific knock-out mice or in presence of blocking antibodies 6,9,13.

<table><tbody><tr><td>5 ml polystyrene round bottom tubes&amp;nbsp;</td><td>Beckton Dickinson</td><td>352058</td><td></td></tr><tr><td>10x CFI Plan Apochromat 0.45&amp;nbsp;&amp;nbsp; DT:4mm&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>20x CFI Plan Apochromat VC 0.75 DT:1mm&amp;nbsp;</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&amp;nbsp;</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

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 °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) ...

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 ...

The overall goal of this intra vital confocal microscopy procedure is to monitor the dynamics of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions in mice. This method can help answer key questions in the immunology field, such as what are the molecular basis of leukocyte migration, recruitment and interaction with the vascular endothelium. The main advantages of this technique are the ability to track and analyze the patrolling of LY 60 low monocytes under statist state conditions, and to follow the recruitment of both monocytes and neutrophils to the same vessel After local inflammatory stimuli demonstrating the procedure will be Stefan ler the technician from the laboratory To prepare a single cell preparation from bone marrow, sterilize the hind legs of an eight to 12 week old mouse with 70%ethanol, and then use a scalpel to harvest the femurs and tibia is when all of the bones have been disconnected.Rinse the legs with 90%ethanol and transfer the bones into a culture dish filled with PBS. Then in a culture hood, use the scalpel to clean the femurs and tibia and to separate them at the knee joint. Next, cut off the ends of the bones and use a one milliliter syringe built with preparation medium equipped with a 23 gauge needle to flush the marrow from each bone into a 50 milliliter conical tube.Disrupt the cell aggregates by passing them back through the needle tip, and then bring the total volume of the cell suspension up to 25 milliliters with fresh preparation, medium spin down the cells, then resus. Suspend the palate in one milliliter of red blood cell lysis buffer, and transfer the cells to a 15 milliliter conical tube on ice. After 30 seconds, stop the lysis with 10 milliliters of preparation.Medium.Spin down the cells again and Resus suspend the pellet in. Two more milliliters of preparation medium to enrich the neutrophils by negative selection. First, incubate the bone marrow cells with 150 microliters of mouse neutrophil enrichment cocktail on ice for 10 minutes.At the end of the incubation at 10 milliliters of phenol red free dmem. Invert the tube one time and spin down the cells. Re suspend the pellet in 1.75 milliliters of preparation medium, and transfer the cells into a five milliliter polystyrene round bottom tube.Then incubate the cells with 250 microliters of biotin selection cocktail on ice for 10 minutes. At the end of the incubation, we suspend the magnetic particles from the cell isolation kit with 30 seconds of vortexing. Then incubate the cells with 500 microliters of the particles on ice for 10 minutes.At the end of the incubation, place the tube into the magnet. After three minutes, invert the magnet into a new five milliliter polystyrene round bottom tube. To collect the unlabeled cells, place the tube of unlabeled cells into the magnet.After three minutes, invert the magnet into a 15 milliliter conical tube to transfer the unlabeled cells leaving the last drop in the tube. Then bring the volume up to five milliliters with fresh phenol red free dm. Now add half a microliter of the appropriate cytoplasmic dye such as the cell tracker orange used here to the cells at 37 degrees Celsius.After two minutes, a 37 degrees Celsius, wash the cells with 2.5 milliliters of preparation medium and resuspend the pellet in one milliliter of preparation medium in a 1.5 milliliter tube, count the cells and then incubate them for five minutes at 37 degrees Celsius. After spinning down the cells, we suspend the pellet in one milliliter of PBS followed by another centrifugation while the cells are spinning. Use oil to attach a 35 millimeter glass cover slip to a 10 centimeter tissue culture dish with a 30 millimeter diameter hole.Then resuspend the pellet at one times 10 to the seven cells per 100 microliters of PBS and place the cells on ice as soon after labeling as possible. Inject the labeled neutrophil cells intravenously into an anesthetized eight to 12 week old CX three CR one GFP mouse, and place the mouse on a heating pad. Then immediately apply ointment to the animal's eyes and use scissors to open the abdominal skin, exposing the peritoneum, incise the peritoneum with the scissors to expose the intestine.Then administer 200 microliters of 37 degrees Celsius PBS to the peritoneal cavity and turn the mouse face down over the tissue culture dish to remove the intestines from the peritoneal cavity with gentle pressure. Then using sterile cotton buds carefully droll the intestines onto the cover slip to expose the mesenteric vessels and immobilize the tissue with pieces of tissue paper wet with 37 degrees Celsius PBS to decrease the movements resulting from peristalsis. It's very important to take your time to carefully immobilize the intestine with PBS weighted tissues.Otherwise movements resulting from peristaltic during image acquisition will ruin the experiment. Next, transfer the tissue culture dish and mouse into a 37 degree Celsius thermostat chamber on a custom made trace stage of an inverted microscope and administer a second round of anesthetics intramuscularly to the hind leg. Now set the appropriate lasers to the lowest power necessary to avoid laser induced phototoxicity and allow the mouse to rest for 30 minutes.When the animal has recovered, record the first movie for 30 minutes under steady state conditions. Then to initiate the blood vessel inflammation, dispense a 20 microliter drop of PBS containing 100 micrograms of the TLR two TLR one agonist PAM three CSK four directly onto the imaged vessels. Finally acquire a second movie taking care to control the focus after addition of the agonist as the inflammation induces changes in the endothelium width, altering the focus of the Z axis here, the different steps of image processing for the first time point of a movie acquired under steady state conditions, as just demonstrated, are shown in this movie.The patrolling of green LASIK cilo monocytes under steady state conditions can be visualized with red neutrophils observed circulating in the bloodstream.Here. The same field of interest is shown 90 minutes after the addition of PAM three CSK four, resulting in the massive recruitment of neutrophils and monocytes to the endothelial wall, which the monocytes scan meticulously using the appropriate software, the exact paths of the monocytes and neutrophils. Patrolling the endothelium can be tracked before and after the initiation of the inflammation.It is important to note that a loss of focus on the Z axis or an XY displacement can occur as a consequence of intestinal movements ruining the experiment. This technique can be further modified by using different genetically modified mice models or injecting blocking antibodies or pharmaceutical drugs to determine the role of specific molecules of interest in leukocyte behavior. After watching this video, you should have a good understanding of how to monitor the dynamics of saturating leukocytes in mesenteric veins.

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Research

JoVE Journal

Immunology and Infection

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.

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

In this video, we demonstrate the real-time visualization of blood cell-endothelial interactions in the mesenterial veins of a mouse model injected intraperitoneally with lipopolysaccharide.

Intravital Microscopy to Analyze Blood Cell-Endothelial Interactions in an Inflammation Mouse Model

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.

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

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 of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in 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.

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

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

We describe a method to visualize GFP-labeled γδ IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions.

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

Learn intravital confocal microscopy to monitor neutrophil and monocyte dynamics in mouse mesenteric veins under steady state and inflammatory conditions. This method enables real-time tracking of leukocyte migration and recruitment.

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

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