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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Dermatol. 134 (11), 2680-2684 (2014).</li><li>Egawa, G., Natsuaki, Y., Miyachi, Y., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+epidermal+keratinocytes+and+dermal+vasculatures+using+two-photon+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+2-photon+imaging+of+blood+flow+in+the+neocortex+through+a+cranial+window.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">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 combination of these three methods enables visualization of the motile activities of primed neutrophils (DsRed+/Ly6G+) in inflammatory skin lesions. The migration direction and velocity of the primed neutrophils is further quantified via migratory path tracking. By recording a series of time-lapse videos, the real-time kinetics of primed neutrophil emergence is monitored at inflammatory site and acquisition of DsRed expression by individual neutrophils is also observed in the extravascular space. Thus, the dynamic process of neutrophil priming has been successfully visualized in living animals for the first time using the technology presented here.
This technology is employed in several critical steps. First, skin tissues are the most convenient body parts for intravital imaging. Topical application of OX on the mouse ear allows direct visualization of immune cell behaviors in inflamed skin under confocal microscopy. Second, the mouse must be sedated constantly during imaging which necessitates repeat injections of anesthetic. However, over-sedation may cause mouse die during imaging. To avoid overdose, low doses of anesthetic (a half or a third of the full dose) are recommended for long-term imaging. Third, fluorescence-conjugated neutrophil-specific mAb must be injected intermittently due to the short lifespan of murine neutrophils (half-life of 8-10 hr30). Last, focus drift is a major issue during time-lapse imaging, which could be caused by multiple factors including temperature variations, mouse body movement, ear swelling over time, and vibrations arising from operation of the instrument31. Several methods have been developed to prevent drifting, such as administration of a constant dose of sedation during imaging, fixing the mouse ear in place by taping a glass slide over it on the stage, installing an environmental chamber to provide oxygen and warmth to the sedated organism as well as by manually re-focusing the scope when necessary32,33.
It is equally important to state the limitations of this protocol. First, a slow mouse recovery (12 - 16 hr) after an imaging period 8 hr in length is observed due to continuous sedation, indicating that this protocol may not be suitable for such lengthy experiments. An alternative approach is to use several mice in sequence over the imaging period rather than using only one. Second, a gradual leak of circulating FITC-dextran is observed during imaging over time, which is likely caused by increased permeability of skin blood vessels in the inflammatory state34. Thus, visualization of blood vessels is markedly compromised given reduced fluorescence in circulation at later stages of imaging. Alternatively, one may use fluorescently conjugated lectins such as isolectin B4 to mark blood vessels as these adhere to vascular endothelial cells35.
In addition to visualization of the process of neutrophil priming in skin inflammatory lesions, the technology described here can also be applied in many other experimental settings. In combination with recently developed imaging windows, intravital microscopy delineating the behaviors of primed neutrophils could be assessed in deep tissues and organs including brain, mammary glands, lungs, and abdominal organs36-39. Moreover, visualization of priming/activation of other cell types such as monocytes and macrophages could be achieved using pIL1-DsRed mice along with in vivo labeling via fluorescence-conjugated mAb specific to the cell type of interest. Taken together, not only does this technology provide a fundamental framework for intravital imaging of the priming process of neutrophils in living animals, but it also unveils new approaches for studying the behavior and function of other immune cells in various inflammatory states unfolding in different tissues.

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 height="21">
			<td height="21" style="height:21px;width:196px;">Heparin sodium</td>
			<td style="width:165px;">APP Pharmaceuticals</td>
			<td style="width:139px;">NDC 63323-540-31</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">ACK lysing buffer</td>
			<td style="width:165px;">Lonza</td>
			<td style="width:139px;">10-548E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Fetal bovine serum</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">F0926</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Lipopolysaccharides</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">L4391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">Ketamine hydrochloride</td>
			<td style="width:165px;">Hospira</td>
			<td style="width:139px;">NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td style="width:165px;">LLOYD Laboratory</td>
			<td style="width:139px;">NADA #139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acepromazine</td>
			<td style="width:165px;">Boehringer Ingelheim</td>
			<td style="width:139px;">ANADA 200-361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair-removal cream</td>
			<td style="width:165px;">Church &#38; Dwight</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">A16P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxazolone</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:139px;">E0753</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:196px;">Alexa Fluor 647 anti-mouse Ly6G antibody</td>
			<td>BioLegend</td>
			<td style="width:139px;">127610</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:196px;">U-100 insulin syringe with 28 G needle</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:196px;">FITC-CM-Dextran, 150 Kda</td>
			<td>Sigma-Aldrich</td>
			<td style="width:139px;">74817</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:196px;">Butterfly infusion set (27 G needle)</td>
			<td>BD</td>
			<td style="width:139px;">387312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCalibur cytometer</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellQuest Pro software</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 complexity (SSC) (Figure 1A)29, and DsRed signal is measured in this gated population. Circulating myeloid cells from WT mice minimally express DsRed, while the same cells from pIL1-DsRed mice express distinguishable DsRed signals (Figure 1B). The pIL1-DsRed mice identified by this phenotyping method are used for subsequent experiments. It should be noted that the complete phenotyping process takes only a few hours&#160;&#8212; much shorter than the traditional genotyping methods including tissue harvest, enzymatic digestion, DNA isolation, PCR and electrophoresis.
IL-1&#946; promoter activation has recently been identified as a marker of neutrophil priming22. Thus, the dynamic process of neutrophil priming can be visualized and assessed in living animals using pIL1-DsRed mice. To induce skin inflammation, the skin sensitizer OX is topically applied on the right ear of pIL1-DsRed mice and vehicle alone is applied on the left ear of the same animal. To label neutrophils in living animals, a recently developed protocol is performed by administering a small amount of fluorescently labeled anti-Ly6G mAb immediately before and 8 hr after OX application. To locate blood vessels, FITC-dextran is injected immediately prior to imaging.
At 24 hr after OX application, a large number of DsRed+/Ly6G+ cells are found in the extravascular space (Figure 2A) and represent primed neutrophils. A small number of DsRed/Ly6G+ cells are also found, which most likely represent resting neutrophils. Moreover, a small population of DsRed+/Ly6G- cells are observed, which may comprise inflammatory monocytes and activated macrophages. In a 40 min time-lapse video, Ly6G+ neutrophils emerging within an inflammatory skin lesion exhibit typical amoeba-like crawling movement (Supplemental Movie 1). Cell migratory paths are tracked to compare motile behaviors of the DsRed+/Ly6G+ and DsRed-/Ly6G+ neutrophil populations (Figure 2B). The track plot analyses reveal that both neutrophil populations display &#34;random&#34; migration once the extracellular space has been entered (Figure 2C). Interestingly, DsRed+/Ly6G+ neutrophils exhibit a significantly higher velocity compared with their DsRed-/Ly6G+ counterparts (Figure 2D). This suggests an association between neutrophil priming and accelerated motility in inflammatory lesions.
To monitor the timing of neutrophil priming, a series of time-lapse videos were recorded starting at different times following OX treatment (Supplemental Movies 2 - 4). DsRed-/Ly6G+ cells, the resting neutrophils, become detectable in the extravascular space as early as 8 - 12 hr after OX application and their numbers remain relatively low thereafter (Figure 2E, Supplemental Movies 3, 4). On the contrary, the number of DsRed+/Ly6G+ cells, the primed neutrophils, increase progressively at later time points, beginning at 14 - 16 hr after OX application. Some DsRed-/Ly6G+ cells gradually acquire DsRed signals after recruitment to the extravascular space during this period (Figure 2F, Supplemental Movie 5). At 16 - 20 hr, a majority of the extravascular Ly6G+ neutrophils are considered as &#34;primed&#34; based on their DsRed expression. These observations reveal the magnitude, tempo, and location of neutrophil priming occurring at inflammatory lesions in living animals.
<img alt="Figure 1" src="/files/ftp_upload/54070/54070fig1.jpg" /> 
Figure 1: Phenotyping of pIL1-DsRed Mice. (A) Flow cytometry analyses demonstrate a population of circulating myeloid cells (G1) based on FSC and SSC parameters. (B) Histograms at the FL2 channel show the percentage of DsRed+ cells within G1 gate of the cells from pIL-DsRed mice (red) or from WT mice (blue). Data are representative of at least three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54070/54070fig2.jpg" /> 
Figure 2: Visualization of Motile Activities of DsRed+/Ly6G+ Neutrophils in Inflammatory Skin Lesions. (A) Static confocal microscopy images recorded 24 hr after OX treatment show three leukocyte populations in the extravascular space, i.e., DsRed+/Ly6G+ cells (asterisks), DsRed-/Ly6G+ cells (triangles), and DsRed+/Ly6G- cells (arrows). Bar = 20 &#181;m. DsRed+/Ly6G+ cells (red) and DsRed-/Ly6G+ cells (blue) are compared for migratory paths (B), track plots (C), and velocity (D) from a 40 min time-lapse movie recorded by confocal microscopy starting 24 hr after OX application (Supplemental Movie 1). (D) Bars indicate mean velocity values. *** P &#60;0.001. (E) The numbers of DsRed+/Ly6G+ cells and DsRed-/Ly6G+ cells are counted from a series of time-lapse movies recorded at different time-points after OX application (Supplemental Movies 2 through 4). The data shown are the cell numbers/mm2 (means &#177; SD) calculated from three consecutive images. (F) Static images are created from a time-lapse movie recorded from 11 to 14.5 hr after OX painting (Supplemental Movie 5). The arrow indicates a Ly6G+ cell that acquires DsRed expression in the extravascular space during the observation period. Bar = 50 &#181;m. Images are reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Movie 1: Motile Behavior of DsRed+ Neutrophils in Inflammatory Skin Lesions (at 24 hr after OX painting). Confocal microscopy images show the motility of DsRed+/Ly6G+ cells (pink) and DsRed-/Ly6G+ cells (blue) in the extravascular space. Blood vessels are seen in green. Bar = 20 &#181;m. The video is reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070video1.mov" target="_blank">Please click here to view this video.</a>
Supplemental Movie 2: Motile Behavior of DsRed+ Neutrophils in Inflammatory Skin Lesions (at 4 hr after OX painting). Confocal microscopy images show DsRed+/Ly6G+ cells (pink) and DsRed-/Ly6G+ cells (blue) in the extravascular space. Blood vessels are seen in green. Autofluorescence is present and associated with hair follicles. Bar = 100 &#181;m. The video is reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070video2.mov" target="_blank">Please click here to view this video.</a>
Supplemental Movie 3: Motile Behavior of DsRed+ Neutrophils in Inflammatory Skin Lesions (at 8 hr after OX painting). Confocal microscopy images show DsRed+/Ly6G+ cells (pink) and DsRed-/Ly6G+ cells (blue) in the extravascular space. Blood vessels are seen in green. Autofluorescence is present and associated with hair follicles. Bar = 100 &#181;m. The video is reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070video3.mov" target="_blank">Please click here to view this video.</a>
Supplemental Movie 4: Motile Behavior of DsRed+ Neutrophils in Inflammatory Skin Lesions (at 11 - 19 hr after OX painting). Confocal microscopy images show DsRed+/Ly6G+ cells (pink) and DsRed-/Ly6G+ cells (blue) in the extravascular space. Blood vessels are seen in green. Autofluorescence is present and associated with hair follicles. Bar = 100 &#181;m. The video is reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070video4.mov" target="_blank">Please click here to view this video.</a>
Supplemental Movie 5: Acquisition of DsRed Signals by Ly6G+ Neutrophils. From the time-lapse movie shown in supplemental Video 4, this higher magnification video shows the process in which a Ly6G+ cell (indicated with an arrow) acquires DsRed expression in the extravascular space during the observation period (11-14.5 hr after OX painting). Bar = 50 &#181;m. The video is reproduced from reference 22 with permission (Copyright 2015. The American Association of Immunologists, Inc.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54070/54070video5.mov" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Intravital Imaging of Neutrophil Priming Using IL-1ÃŽÂ² 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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.5K 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

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 ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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 ...

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, ...

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 ...

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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

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. ...