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 ...

<ol>
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G., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+against+neutrophil+LY6G+do+not+inhibit+leukocyte+recruitment+in+mice+in+vivo.">Antibodies against neutrophil LY6G do not inhibit leukocyte recruitment in mice in vivo.</a> Blood. 121 (1), 241-242 (2013).</li><li>Kikushima, K., Kita, S., Higuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+imaging+for+the+in+vivo+tracking+of+high-speed+vesicle+transport+in+mouse+neutrophils.">A non-invasive imaging for the in vivo tracking of high-speed vesicle transport in mouse neutrophils.</a> Sci. 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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 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 complexi...

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

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

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 ...

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

9.5K visualizações.  University of Toledo College of Medicine and Life Sciences. 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....