Name: Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice (Video) | JoVE Journal
Uploaded: 2016-06-22T13:00:00.000+00:00
Duration: 9 min 34 s
Description: Learn intravital imaging of neutrophil priming using IL-1β promoter-driven DsRed reporter mice. This method combines fluorescent reporters, in vivo labeling, and real-time imaging to visualize immune cell behavior in living animals.

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5 minutes

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>Hogan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Focusing+on+the+experiment%5BTitle%5D)+AND+%22Biophotonics.+Int%22%5BJournal%5D)">Focusing on the experiment.</a> Biophotonics. Int. 13, 48-51 (2006).</li>
<li>Kabashima, K., Egawa, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+multiphoton+imaging+of+cutaneous+immune+responses%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">Intravital multiphoton imaging of cutaneous immune responses.</a> J. Invest. 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/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>
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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

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-1β 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 (S...

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 ...

The overall goal of this procedure is to present a technology for monitoring the process of neutrophil priming in living animals. This method can help answer the key questions in the immunology field, such as visualization of the behavior and the function of priming the neutrophils in animal models of rarest diseases and disorders. The main advantage of this technique is that it combines three established methodologies, including fluorescent reporters in viral labeling and intravital imaging.Breed heterozygous pIL1-DsRed mice with wild-type C57Black six mice. At three to four weeks, phenotype their pups. For each pup, prepare a 1.5 milliliter tube with 20 microliters of heparin.To collect blood, hold a pup by its neck scruff and pierce the submandibular vein in the cheek pouch using a 5 millimeter lancet. Only a small puncture is needed. Collect three to five drops of blood per tube.After making the collection, be sure to stop the bleeding and observe the animals for 30 minutes before returning them to the animal facility. To each sample tube, add 500 microliters of red blood cell lysing buffer. Then, vortex the tubes, and incubate them for five to 10 minutes on ice.After the incubation, slowly add 400 to 500 microliters of fetal bovine serum under the cell suspensions in each tube. A clear interface should form between the layers. Next, centrifuge the samples at 1, 500 Gs for five minutes to collect cell pellets.Discard the supernatent and resuspend the cells in 200 microliters of complete RPMI-1640 medium. Then, transfer each suspension to a flow cytometry tube and add 20 microliters of freshly made LPS Working Solution to each tube. Incubate the samples for four hours before performing flow cytometry.To begin, anesthetize the mouse with an intraparetoneal injection of an anesthetic cocktail and confirm adequate sedation with a toe pinch test. Next, apply hair removal cream to the dorsal surface of both ears, wipe it off within a minute, and wash the skin with water. Measure the thickness of the ear where the applications were made using a micrometer.Later, return the mouse in its own cage after it has recovered from the anesthesia, and allow the mouse to rest for at least three days before further testing. After three days, reanesthetize the mouse and measure the ear thickness again to make sure it is not irritated. Then, apply 12.5 microliters of 1.25%oxazolone onto each side of the right ear, and apply 12.5 microliters of the acetone olive oil vehicle solution onto each side of the left ear.As always, observe the mouse recover from the anesthesia. 24 hours later, measure the thickness of the ear again under anesthesia to ensure the oxazolone induced skin inflammation has occurred. To begin, transfer 100 microliters of anti-lyse XG monocolonal antibody into a U100 insulin syringe with a 28 gauge needle.Next, anesthetize a treated mouse as previously described. With the mouse abdomen down, apply gentle downward pressure to the skin dorsal and ventral to an eye to partially protrude the eyeball. Then, carefully place the needle, bevel down, approximately 30 degrees to the medial canthus and carefully insert the needle into the retro orbital sinus.The penetration pressure will be relieved when the needle enters the sinus. Then, eject the content and remove the needle quickly. A small amount of bleeding suggests successful injection.Now, immediately apply 1.25%oxazolone and vehicle solution onto the right and left mouse ears as before. Eight hours later, administer a second dose of the same antibody into each mouse. Also, to visualize the blood vessels, retro orbitally administer 100 microliters of fitc dextran.For this procedure, first anesthetize the mouse as before. Then, apply veterinary ointment to the eyes to prevent dryness during imaging. Next, place a glass coverslip at the center of an imaging stage, and secure the edges of the coverslip with tape.Also, add a drop of PVS to the coverslip. Now, position the mouse's ear over the coverslip, dorsal side down. Next, moisten the ventral surface of the mouse ear with a drop of PVS.Then, mount the tip of the ear with a glass slide on top and secure the slide with tape. Now, turn on a multi laser fluorescence confocal microscope and the related equipment. Turn off the room lights to minimize ambient light exposure.Then, record three dimensional images using the confocal microscope. For time lapse imaging experiments, record three dimensional images every two to four minutes for up to eight hours. All the while, watch for twitching whiskers, which indicates that the mouse is beginning to recover from the anesthesia.In such situations, administer five to 10 microliters of a half dose of anesthetic cocktail via a butterfly needle. pIL-1-DsRed mice were screened for phenotypic DsRed fluorescence produced in peripheral blood leukocytes via flow cytometry. Circulating myeloid cells from wild-type mice minimally express DsRed, while the same cells from pIL1-DsRed mice express distinguishable DsRed signals.IL-1 beta promoter activation is a marker of neutrophil priming, and can be best visualized in living pIL-1DsRed mice. Next, a skin sensitizer, oxazolone, was topically applied to the right ear. Later, the neutrophils were labeled before and after a second application of the oxazolone sensitizer via retro orbital injection.Likewise, fitc dextran was injected to locate blood vessels. Many double labeled cells were found in the extravascular space, which represent primed neutrophils. A small number of cells were only labeled by the lyse XG antibody, which were probably resting neutrophils.Moreover, a small population of DsRed positive cells were observed, which may comprise inflammatory monocytes and activated macrophage. Cell migratory paths were tracked to compare the two neutrophil populations. Both neutrophil populations showed a random migration once the extracellular space had been entered.Interestingly, the putatively primed neutrophils exhibited a significantly higher velocity compared with their putatively resting counterparts. After its development, this technique paved the way for researchers in the field of immunology. Two explore the behavior and the function of many immune cells in various inflammatory states involving different t-cells.After watching this video, you should have a good understanding of how to monitor the dynamic process of neutrophil priming in living animals.

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

Research

JoVE Journal

Immunology and Infection

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.

Non-invasive Imaging of Leukocyte Homing and Migration 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.

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

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.

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

This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen—a hapten in an oil/acetone solution—is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period.

A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

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

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

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

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

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.

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

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.

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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.

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

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

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

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

Learn intravital imaging of neutrophil priming using IL-1β promoter-driven DsRed reporter mice. This method combines fluorescent reporters, in vivo labeling, and real-time imaging to visualize immune cell behavior in living animals.

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

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