The authors have no acknowledgements.

All animal experiments are performed in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of the University of Toledo.
1. Phenotyping of pIL1-DsRed Mice
NOTE: Offspring are generated by breeding heterozygous pIL1-DsRed mice with wild-type (WT) C57BL6 mice. Three to four week old pups are considered ready for phenotyping. Submandibular bleeding of mice follows an established protocol with minor modifications23.
<ol>
	<li>Isolation of White Blood Cells from the Whole Blood of Mouse Pups
	<ol>
		<li>Add 20 &#181;l of heparin to each 1.5 ml microcentrifuge tube.</li>
		<li>Acquire one pup from the cage. Record the identification tag of the pup. It is not required to anesthetize pups before blood collection from the submandibular vein.</li>
		<li>Hold pup by neck scruff and pierce the submandibular vein in the cheek pouch using a 5 mm lancet. Apply adequate force to create a small puncture, so that drops of blood exude from the point of penetration. Use a new needle for each pup.</li>
		<li>Collect 3 - 5 drops of blood per pup in a heparin-containing microcentrifuge tube. Clean the puncture site and apply pressure to facilitate hemostasis.</li>
		<li>Put pup in new cage and observe for 30 min before returning the cage to the animal facility.</li>
		<li>Collect blood from adult mice of a known phenotype as positive and negative controls.</li>
		<li>Add 500 &#181;l of red blood cell lysing buffer to each tube, vortex tubes, and incubate for 5 - 10 min on ice.</li>
		<li>Slowly add 400 - 500 &#181;l of fetal bovine serum (FBS) underneath the cell suspensions in each tube. A clear interface can be observed between the upper-layer cell suspension and the lower-layer of FBS.</li>
		<li>Cap tubes and centrifuge samples at 1,500&#160;&#215; g for 5 min.</li>
		<li>Repeat steps 1.1.7 - 1.1.9 to further remove red blood cells. Do not repeat if lysis is sufficient the first time. The pellet should be difficult to discern after centrifugation.</li>
	</ol>
	</li>
	<li>Lipopolysaccharide (LPS) Stimulation and Culture
	<ol>
		<li>Resuspend cells in 200 &#181;l of complete Roswell Park Memorial Institute (RPMI) 1640 medium.</li>
		<li>Transfer the cell suspension to a flow cytometry tube.</li>
		<li>Make LPS working solution by mixing 10 &#181;l of LPS stock solution (1 mg/ml) with 990 &#181;l of complete RPMI 1640 medium.</li>
		<li>Add 20 &#181;l of 10 &#181;g/ml LPS working solution to 200 &#181;l&#160;of cell suspension.</li>
		<li>Cap tubes loosely and incubate samples for 4 hr at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Flow Cytometry Analysis
	<ol>
		<li>Open the data acquisition program for flow cytometry. Set up acquisition template prior to sample acquisition.</li>
		<li>Click the Dot-Plot tool in the tool palette. Draw a forward scatter (FSC) vs side scatter (SSC) plot. Set the FSC and SSC voltage parameters to linear scale.</li>
		<li>Select the lowest thresholds of FSC and SSC allowed by the cytometer. Apply thresholds 200 and 50 for FSC and SSC, respectively.</li>
		<li>Within the FSC vs SSC plot, draw a gate G1 which includes monocytes, granulocytes, and dendritic cells, and excludes debris, red blood cells and lymphocytes.</li>
		<li>Click the Histogram tool in the tool palette. Draw a FL2 (red fluorescence channel) histogram. Use a negative control sample to set the baseline of FL2 signals and a positive control sample to draw a gate to include all DsRed positive events.</li>
		<li>On the Fluidics Control panel of the cytometer, set the fluid mode to &#34;RUN&#34; and the flow rate to &#34;HI&#34; (approximately 60 &#181;l/min). Acquire 10,000 events for each sample.</li>
		<li>Determine pup phenotype by measuring the percentage of DsRed positive cells from the G1 gate.</li>
	</ol>
	</li>
</ol>
2. Induction of Skin Inflammation in pIL1-DsRed Mice
<ol>
	<li>Anesthetize mouse by intraperitoneal (i.p.) injection of an anesthetic cocktail containing 100 mg/kg ketamine, 10 mg/kg xylazine, and 1 mg/kg acepromazine. Adequately anesthetized mice do not show hind paw withdrawal to toe pinch.</li>
	<li>Apply hair-removal cream to the dorsal surface of both ears of the mouse. Wait 30 sec to 1 min. Wipe ear surface with water-wetted cotton and allow to air-dry.</li>
	<li>Measure the ear thickness using micrometer and replace mouse in a separate cage. Observe until recovery from anesthesia (~ 15 - 30 min). To resolve skin inflammation induced by the hair removal product, allow mouse to rest for 3 days before further experimentation is carried out.</li>
	<li>Prepare 1.25% (w/v) OX by dissolving 16.7 mg of OX in 1 ml acetone and mixing 750 &#181;l of OX/acetone solution with 250 &#181;l of olive oil. Prepare vehicle solution by mixing 750 &#181;l of acetone with 250 &#181;l of olive oil.</li>
	<li>Re-anesthetize mouse by i.p. injection of anesthetic cocktail (2.1).&#160;Measure the ear thickness as before.</li>
	<li>Apply 12.5 &#181;l of 1.25% OX onto each side of right ear. Apply 12.5 &#181;l of acetone/olive oil vehicle solution onto each side of left ear.</li>
	<li>Keep mouse separate from cage-mates until fully recovered to prevent insult to its ears by other mice, then replace the mouse in its original cage and return to animal housing facility. 
	NOTE: Hair removal may result in minor skin inflammation following by change of ear thickness. This minor inflammation will be resolved 3 days later confirmed by normal ear thickness. After topical application for 24 hr, OX-treated ears show significant (p &#60; 0.01) swelling compared vehicle solution alone-treated ears.</li>
</ol>
3. Labeling of Neutrophils in pIL1-DsRed Mice
NOTE: In vivo labeling of neutrophils by low dose fluorescence-conjugated neutrophil-specific mAb follows a recently developed protocol19. Retro-orbital injections are performed according to an established protocol with a few modifications24.
<ol>
	<li>Prepare anti-Ly6G mAb working solution by diluting 10 &#181;l of 0.5 mg/ml Alexa Fluor 647-conjugated anti-Ly6G mAb (clone 1A8) in 90 &#181;l of phosphate buffered saline (PBS).</li>
	<li>Transfer 100 &#181;l of anti-Ly6G mAb working solution (50 &#181;g/ml) into a U-100 insulin syringe with 28 gauge needle.</li>
	<li>Anesthetize an adult pIL1-DsRed mouse by i.p. injection of the previously described anesthetic cocktail (2.1). Place the anesthetized mouse abdomen down on a clean working board.</li>
	<li>Apply gentle downward pressure to the skin dorsal and ventral to one mouse eye to partially protrude the eyeball from the socket.</li>
	<li>Carefully place the needle, bevel down, at an angle of approximately 30&#176; at the medial canthus into the retro-orbital sinus. 
	NOTE: The operator can feel pressure, penetration and relieved resistance once the needle inserts into the sinus.</li>
	<li>Slowly and smoothly inject 100 &#181;l of anti-Ly6G mAb working solution (5 &#181;g mAb/mouse/injection) into the mouse. Remove the needle quickly. A small amount of bleeding suggests a successful injection.</li>
	<li>Immediately apply 1.25% OX and vehicle solution onto the right and left mouse ears.</li>
	<li>After 8 hr, administer 100 &#181;l of freshly prepared anti-Ly6G mAb working solution (5 &#181;g mAb/mouse/injection) via retro-orbital injection into the same mouse.</li>
	<li>To visualize the blood vessels, immediately before imaging, administer 100 &#181;l of 30 mg/ml fluorescein isothiocyanate (FITC)-conjugated dextran (150 kDa) through retro-orbital injection.</li>
</ol>
4. Intravital Imaging of Neutrophil Priming in pIL-1-DsRed Mice
<ol>
	<li>Anesthetize mouse with i.p. injection of anesthetic cocktail (2.1).&#160;Apply veterinary ointment to mouse eyes to prevent dryness while under anesthesia for imaging.
	<ol>
		<li>Prepare 1 ml half-dose anesthetic cocktail by diluting 500 &#181;l of original anesthetic cocktail with an equal volume of PBS.</li>
		<li>Transfer half-dose anesthetic cocktail to 1 ml syringe with a butterfly 27 gauge needle.</li>
		<li>Insert needle of butterfly into mouse abdomen intraperitoneally and fix the butterfly wing to the abdomen by taping.</li>
	</ol>
	</li>
	<li>Place a glass coverslip in the center of the imaging stage. Tape the edges of the coverslip to hold it in place.</li>
	<li>Place a drop of PBS on the ventral surface of the mouse ear as well as on the coverslip.</li>
	<li>Position anesthetized mouse with its ear over the coverslip. Mount the tip of the ear, dorsal&#160;side down, by sandwiching the ear between the coverslip and a glass slide, also held in place via tape.</li>
	<li>Turn on a multi-laser fluorescence confocal microscope and all the related equipment. Turn off room lights to minimize ambient light exposure.</li>
	<li>Open the image acquisition software. Select the laser channels for detection of fluorescent dyes in the Dye List menu.</li>
	<li>Adjust the focus on the inflamed ear skin by observation of green signals from the blood vessels and red signals from the DsRed+ cells through eyepiece.</li>
	<li>Perform a fast scan with scan speed of 2 &#181;s/pixel and resolution of 512 &#215; 512 pixels. Select a pseudocolor for each channel in the Live View menu. Adjust the focus knob to set the end and start location of scanning.</li>
	<li>Perform slow scans with scan speed of 4 - 8 &#181;sec/pixel and resolution of 800 &#215; 800 or 1,024 &#215; 1,024 pixels. Adjust the high voltage, gain, and offset on each individual channel to maximize the intensity of signals while avoiding saturation (red pixels). There should be only a few red pixels in each channel.</li>
	<li>Create three-dimensional image sets by scanning the ear skin beginning superficially in the stratum corneum and progressing downward with x, y, z volumes of 317 &#181;m &#215; 317&#160;&#181;m &#215; 30 &#181;m (40X objective), 635 &#181;m &#215; 635&#160;&#181;m &#215; 30 &#181;m (20X objective), or 1,270&#160;&#181;m &#215; 1,270 &#181;m &#215; 50 &#181;m (10X objective) at 2 &#181;m z-steps. 
	NOTE: The stratum corneum is the outermost layer of epidermis and can be readily localized based on its strong autofluorescence signal.</li>
	<li>In time-lapse imaging experiments, record three-dimensional images every 2 or 4 min for up to 8 hr.</li>
	<li>Intermittently, as mouse begins to recover from anesthesia, noted based on observation of twitching whiskers, administer 5 - 10 &#181;l of half-dose anesthesia cocktail through butterfly needle. 
	NOTE: Be cautious of the anesthesia dose - overdose may cause mouse death.&#160;Alternatively, inhalation anesthesia in a jar could be applied to the mouse for short-term anesthesia and a nose cone could be used for long-term imaging.&#160;</li>
	<li>Remove the anesthetized mouse from the stage when imaging is complete. Detach tape and remove butterfly needle from the mouse&#39;s abdomen. Return the mouse to a separate cage in the animal housing facility. 
	NOTE: For short-term imaging (&#60;1 hr), mice fully recover from anesthesia quickly in 1 - 3 hr. For long-term imaging (~ 8 hr), mice recover slowly, with a typical recovery period of 12 - 16 hr. Thus, oral water administration is recommended at least several times during the recovery period to prevent severe dehydration.</li>
	<li>Process imaging datasets, track individual cells, generate migratory paths, and calculate the directionality and velocity of cell migration using image analysis software25.</li>
</ol>

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<li>Egawa, G., Natsuaki, Y., Miyachi, Y., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+imaging+of+epidermal+keratinocytes+and+dermal+vasculatures+using+two-photon+microscopy%5BTitle%5D)+AND+%22J.+Dermatol.+Sci%22%5BJournal%5D)">Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy.</a> J. Dermatol. Sci. 70 (2), 143-145 (2013).</li>
<li>Kedrin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat. Methods. 5 (12), 1019-1021 (2008).</li>
<li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+2-photon+imaging+of+blood+flow+in+the+neocortex+through+a+cranial+window%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">A method for 2-photon imaging of blood flow in the neocortex through a cranial window.</a> J. Vis. Exp. (12), (2008).</li>
<li>Ritsma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Surgical implantation of an abdominal imaging window for intravital microscopy.</a> Nat. Protoc. 8 (3), 583-594 (2013).</li>
<li>Looney, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat. Methods. 8 (1), 91-96 (2011).</li>
</ol>

The authors have no financial conflicts of interest.

The aim of this study is to develop a technology for monitoring the process of neutrophil priming in living animals, which has not yet been fulfilled by the currently available techniques. To achieve this goal, three established methodologies are performed: 1) induction of skin inflammation in IL-1&#946; promoter-driven DsRed reporter mice as a measure of priming, 2) in vivo labeling of neutrophils with low doses of fluorescence-conjugated anti-Ly6G mAb, and 3) intravital confocal microscopy imaging. The combina...

Neutrophils are the most abundant and short-lived leukocytes in circulation. They are rapidly recruited to the sites of infection or injury, where they serve as professional phagocytes through release of reactive oxygen and nitrogen intermediates along with granules containing antimicrobial peptides and proteases1. During their recruitment, neutrophils are &#34;primed&#34; by various agents including microbial products, chemoattractants, and inflammatory cytokines, resulting in markedly enhanced phagocyte functionality upon arrival at a site of inflammation2. The mechanisms of neutrophil priming have been extensively studied in vitro3,4; however, dynamic monitoring of the process in vivo has not been possible to date.
Recently, intravital imaging has become an important technique for visualizing and quantifying the cellular dynamics of biological processes in living organisms. Intravital imaging can be performed via conventional one-photon excitation microscopy (e.g., confocal) or multiphoton microscopy approaches5. Over time, substantial improvements have been achieved in this technique enabling increased image resolution, improved imaging depth, decreased tissue photodamage, and enhanced image stabilization6,7. Given its unique ability to enable dynamic visualization of cellular migration and interaction over time, intravital microscopy has been extensively applied to diverse areas of study in immunology8. Intravital imaging enables immunologists to better understand and contextualize immune responses at both the cellular and molecular level in living animal models.
Recent advances in transgenic as well as knock-in reporter mice have provided useful tools for monitoring the dynamic behaviors of neutrophils in living animals. Lysozyme M promoter-driven enhanced green fluorescent protein knock-in mice have been broadly used to characterize motility of neutrophils, monocytes, and macrophages during various inflammatory processes including extravasation, bacterial infection, and sterile inflammation9-15. Further, transgenic mice expressing a cytoplasmic fluorescence resonance energy transfer biosensor have been employed in studying the activities of neutrophil extracellular-regulated mitogen kinase and protein kinase A within inflamed intestine16. A murine model with high specificity for fluorescence expression in neutrophils is the Catchup knock-in mouse, which produces Cre recombinase as well as the fluorescent protein tdTomato, which itself is coupled to expression of lymphocyte antigen 6G (Ly6G)17. Visualization of Ly6G-deficient neutrophils via this model has demonstrated that these cells exert normal functionality in a variety of sterile or infectious in vivo inflammatory contexts. Transgenic mice expressing the DsRed fluorescent protein gene under the control of the mouse interleuikin-1&#946; (IL-1&#946;) promoter (pIL1-DsRed) have been utilized to visualize the motile behaviors of IL-1&#946; producing cells &#8212; believed to include neutrophils, inflammatory monocytes, and activated macrophages &#8212; emerging in inflamed skin18.
In vivo labeling can serve as an alternative approach for tracing the cellular and molecular behaviors of neutrophils in inflamed tissues. After intravenous injection of low doses of fluorescently labeled anti-Gr-1 monoclonal antibody (mAb), the recruitment cascade of Gr-1+ neutrophils has been visualized in mouse skin lesions infected with Staphylococcus aureus19. In vivo administration of conjugates containing streptavidin-conjugated 705 nm quantum dots and biotinylated anti-Ly6G mAb specifically label circulating neutrophils20. Moreover, endocytosis of such conjugates into neutrophil vesicles allows tracking of high-speed vesicle transport in neutrophils migrating into the interstitium. In vivo labeling with fluorescence-conjugated antibodies against P-selectin glycoprotein ligand-1 (PSGL-1), L-selectin (CD62L), integrin &#945;M (CD11b) and chemokine (C-X-C motif) receptor 2 (CXCR2) in a TNF&#945;-induced inflammatory model has elucidated the regulatory mechanisms at play during early inflammation21. Polarized neutrophils protrude PSGL-1-enriched uropods to interact with CD62L present on activated platelets, resulting in the redistribution of CD11b and CXCR2, receptors that drive neutrophil migration and initiate the inflammation.
IL-1&#946; is one of the signature genes that is elevated in primed neutrophils22. In pIL1-DsRed reporter mice, DsRed fluorescence signals (i.e., activation of IL-1&#946; promoter) positively correlate with IL-1&#946; mRNA expression and IL-1&#946; protein production.18 To monitor the process of neutrophil priming, an intravital microscopy method was developed involving induction of skin inflammation with oxazolone (OX) in the pIL1-DsRed mouse model following in vivo labeling of neutrophils with fluorescence-conjugated anti-Ly6G mAb. Via this model, it is possible to study the behavior and function of primed neutrophils in animal models of various diseases and disorders.

<table><tbody><tr><td>Heparin sodium</td><td>APP Pharmaceuticals</td><td>NDC 63323-540-31</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Lonza</td><td>10-548E</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich</td><td>F0926</td><td></td></tr><tr><td>Lipopolysaccharides</td><td>Sigma-Aldrich</td><td>L4391</td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Hospira</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>Xylazine</td><td>LLOYD Laboratory</td><td>NADA #139-236</td><td></td></tr><tr><td>Acepromazine</td><td>Boehringer Ingelheim</td><td>ANADA 200-361</td><td></td></tr><tr><td>Hair-removal cream</td><td>Church &amp;amp; Dwight</td><td></td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A16P4</td><td></td></tr><tr><td>Oxazolone</td><td>Sigma-Aldrich</td><td>E0753</td><td></td></tr><tr><td>Alexa Fluor 647 anti-mouse Ly6G antibody</td><td>BioLegend</td><td>127610</td><td></td></tr><tr><td>U-100 insulin syringe with 28 G needle</td><td>BD</td><td>329461</td><td></td></tr><tr><td>FITC-CM-Dextran, 150 kDa</td><td>Sigma-Aldrich</td><td>74817</td><td></td></tr><tr><td>Butterfly infusion set (27 G needle)</td><td>BD</td><td>387312</td><td></td></tr><tr><td>FACSCalibur cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>CellQuest Pro software</td><td>BD</td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Metamorph Software</td><td>Universal Imaging</td><td></td><td></td></tr></tbody></table>

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.

Screening of pIL1-DsRed mice is performed based on the phenotypic DsRed fluorescence signal produced by their peripheral blood leukocytes using flow cytometry. LPS stimulation is known to induce IL-1&#946; production in myeloid cells including neutrophils, monocytes, and dendritic cells26-28. Thus, isolated leukocytes are incubated with LPS for 4 hr prior to flow cytometry analysis. Next, gating is set for circulating myeloid cells based on cell size (FSC) and internal complexi...

Watch this Scientific Journal Video about Intravital Imaging of Neutrophil Priming Using IL-1Î² Promoter-driven DsRed Reporter Mice at JoVE.com

Intravital Imaging of Neutrophil Priming Using IL-1&#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 ...

intravital-imaging-neutrophil-priming-using-il-1-promoter-driven

Research

JoVE Journal

Immunology and Infection

9.6K Views.  University of Toledo College of Medicine and Life Sciences. 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. 

Video: Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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 ...

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

Biology

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

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

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.

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

Monitoring Neutrophil Recruitment Dynamics and Bacterial Burden at the Infection Site in a Murine Model

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo imaging to study the neutrophil recruitment dynamics at an infection site in an immunocompromised, transgenic mouse with GFP-expressing neutrophils. It involves injecting luminescent bacteria to cause an infection. This leads to neutrophil recruitment to the infection site, whose tracking is done by imaging.

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

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