Name: intravital imaging of neutrophil priming using il-1&#946; promoter-driven dsred reporter mice
Uploaded: 2016-06-22T13:00:00
Description: Watch this Scientific Journal Video about Intravital Imaging of Neutrophil Priming Using IL-1Î² Promoter-driven DsRed Reporter Mice at JoVE.com

The authors have no acknowledgements.

يتم تنفيذ جميع التجارب على الحيوانات وفقا للمبادئ التوجيهية المعاهد الوطنية للصحة التي وافقت عليها لجنة رعاية واستخدام الحيوان المؤسسي من جامعة توليدو. 1. Phenotyping من pIL1-عن dsRed الفئران يتم إنشاؤ?...

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والهدف من هذه الدراسة هو تطوير التكنولوجيا لرصد عملية فتيلة العدلات في الحيوانات الحية، التي لم يتم الوفاء بها من خلال التقنيات المتاحة حاليا. ولتحقيق هذا الهدف، نفذت ثلاث منهجيات محددة: 1) تحريض التهاب الجلد في IL-1β عن dsRed الفئران مراسل يحركها المروج كمقياس للفتيلة، 2...

العدلات هي الكريات البيض الأكثر وفرة وقصيرة الأجل في الدورة الدموية. كانوا يجندون بسرعة إلى مواقع الإصابة أو الإصابات، حيث أنها تخدم البالعات كما المهنية من خلال الافراج عن الأوكسجين والنيتروجين وسيطة رد الفعل جنبا إلى جنب مع حبيبات تحتوي على الببتيدات المضادة للميكروبات والبروتياز 1. خلال تجنيدهم، العدلات هي &quot;تستعد&quot; من قبل مختلف وكلاء بما في ذلك المنتجات الميكروبية، chemoattractants، والسيتوكينات الالتهابية، مما أدى إلى تعزيز ملحوظ وظيفة بلعمية لدى وصوله إلى موقع الالتهاب 2. وقد تم دراسة آليات فتيلة العدلات على نطاق واسع في المختبر 3،4. ومع ذلك، لم يكن رصد دينامية العملية المجراة يمكن حتى الآن. في الآونة الأخيرة، أصبح التصوير حيوي داخلي تقنية هامة لتصور وقياس ديناميكية الخلوية من العمليات البيولوجية في الكائنات الحية. Intraviتل التصوير لا يمكن أن يؤديها من خلال التقليدية-فوتون واحد الإثارة المجهر (على سبيل المثال، متحد) أو نهج multiphoton المجهري 5. مع مرور الوقت، وقد تم تحقيق تحسينات كبيرة في هذه التقنية تمكن زيادة دقة وضوح الصورة، وتحسين عمق التصوير، وانخفاض photodamage الأنسجة، وتعزيز 6،7 تثبيت الصورة. ونظرا إلى قدرة فريدة من نوعها لتمكين التصور الدينامي للهجرة الخلوية والتفاعل مع مرور الوقت، وقد تم تطبيق intravital المجهري نطاق واسع لمختلف مجالات الدراسة في علم المناعة 8. التصوير حيوي داخلي يمكن المناعة لفهم أفضل وتأطير الاستجابات المناعية في كل من المستوى الخلوي والجزيئي في العيش النماذج الحيوانية. وقد وفرت التطورات الحديثة في مجال المعدلة وراثيا وكذلك تدق في الفئران مراسل أدوات مفيدة لرصد السلوكيات الحيوية العدلات في الحيوانات الحية. الليزوزيم M مدفوعة المروج تعزيز البروتين الفلوري الأخضر تدق فيوقد استخدمت الفئران على نطاق واسع لوصف الحركة من العدلات، وحيدات، والضامة خلال عمليات الالتهابات المختلفة بما في ذلك التسرب، والعدوى البكتيرية، والتهاب عقيم 9-15. وعلاوة على ذلك، وقد استخدمت الفئران المعدلة وراثيا معربا عن حشوية مضان بالرنين نقل الطاقة جهاز الاستشعار البيولوجي في دراسة أنشطة المتعادلة كيناز mitogen خارج الخلية الخاضعة للوائح وبروتين كيناز وداخل الأمعاء الملتهبة 16. نموذج الفئران مع خصوصية عالية للتعبير عن مضان في العدلات هو الضربة القاضية في الماوس صلصة الطماطم، التي تنتج لجنة المساواة العرقية recombinase فضلا عن tdTomato بروتين فلوري، الذي يقترن نفسها في التعبير من اللمفاويات مستضد 6G (Ly6G) 17. وقد أثبتت التصور العدلات Ly6G التي تعاني من نقص عبر هذا النموذج أن هذه الخلايا تمارس وظيفة عادية في مجموعة متنوعة من السياقات التهابات الجسم الحي معقمة أو المعدية في. الفئران المعدلة وراثيا معربا عن عن dsRed الفلورسنت صrotein الجينات تحت سيطرة الماوس interleuikin-1β استخدمت (IL-1β) المروج (pIL1-عن dsRed) لتصور السلوكيات متحركة من الخلايا المنتجة IL-1β - يعتقد انها تضم ​​العدلات، وحيدات التهابات، وتفعيل الضامة - الناشئة في التهاب الجلد 18. في الجسم الحي العلامات يمكن أن تكون نهجا بديلا لتتبع سلوكيات الخلوية والجزيئية العدلات في الأنسجة الملتهبة. بعد الحقن في الوريد بجرعات منخفضة من fluorescently المسمى مكافحة GR-1 ريتوكسيماب (ماب)، شلال توظيف GR-1 + العدلات وقد تصور في الآفات الجلدية الماوس المصابين المكورات العنقودية الذهبية (19). وفي الجسم الحي إدارة تقارن تحتوي على streptavidin- مترافق 705 نقاط الكم نانومتر والمعقدة البيروكسيديز مكافحة Ly6G ماب تسمية تحديدا العدلات المتداولة 20. وعلاوة على ذلك، الإلتقام هذه تقارن في neutrophالحويصلات ايل تتيح تتبع النقل حويصلة عالية السرعة في العدلات المهاجرة في الخلالي. المجراة وضع العلامات مع الأجسام المضادة مضان مترافق ضد ف selectin بروتين سكري يجند-1 (PSGL-1)، L-selectin (CD62L)، إنتغرين αM (CD11b ) وchemokine (CXC عزر) مستقبلات 2 (CXCR2) في نموذج التهابات الناجمة عن TNFα وإجمال الآليات التنظيمية في اللعب أثناء التهاب في وقت مبكر 21. العدلات الاستقطاب تبرز uropods PSGL-1-التخصيب للتفاعل مع CD62L الحاضر على الصفائح الدموية تفعيلها، مما أدى إلى إعادة توزيع CD11b وCXCR2، المستقبلات التي تدفع الهجرة العدلة والشروع في التهاب. IL-1β هي واحدة من الجينات التوقيع الذي ارتقى في العدلات معبي 22. في الفئران مراسل pIL1-عن dsRed، وإشارات مضان عن dsRed (أي.، تفعيل المروج IL-1β) ترتبط بشكل إيجابي مع IL-1β التعبير مرنا وإنتاج بروتين IL-1β. &lt;سوب&gt; 18 لمراقبة عملية فتيلة العدلات، وضعت طريقة intravital المجهري تنطوي على تحريض التهاب الجلد مع oxazolone (OX) في نموذج الفأر pIL1-عن dsRed التالية في الجسم الحي وسم العدلات مع مضان مترافق مكافحة Ly6G ماب. عبر هذا النموذج، فمن الممكن لدراسة سلوك وظيفة من العدلات معبي في النماذج الحيوانية من مختلف الأمراض والاضطرابات.

<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

العدلات هي الكريات البيض الأكثر وفرة في الإنسان الدورة الدموية وجندت بسرعة إلى مواقع التهابات. فتيلة هو الحدث الحاسم الذي يعزز وظيفة أكلة العدلات. على الرغم من أن دراسات مستفيضة وقد كشفت وجود وأهمية فتيلة العدلات خلال العدوى والإصابة، يعني من تصور هذه العملية في الجسم الحي كانت غير متوفرة. بروتوكول المقدمة يتيح رصد عملية ديناميكية من العدلات فتيلة في الحيوانات التي تعيش من خلال الجمع بين ثلاث منهجيات: 1) عن dsRed إشارة مراسل - تستخدم كمقياس للفتيلة 2) في الجسم الحي العدلات وسم - تحقيق ذلك عن طريق حقن مضان مترافق مكافحة اللمفاويات مستضد 6G (Ly6G) الأجسام المضادة وحيدة النسيلة (ماب) و 3) التصوير متحد البؤر حيوي داخلي. وتشارك العديد من الخطوات الهامة في هذا البروتوكول: التي يسببها oxazolone الماوس التهاب الجلد الأذن، والتخدير المناسب للحيوانات، تكرار الحقن المضادة للLy6G ماب، والسابقention التركيز الانجراف أثناء التصوير. على الرغم من أن لوحظ عدد قليل من القيود، مثل الحد من وقت التصوير المستمر (~ 8 ساعات) في الماوس واحد وتسرب ثيوسيانات-ديكستران فلوريسئين من الأوعية الدموية في حالة التهابات، ويوفر هذا البروتوكول إطارا أساسيا للتصوير حيوي داخلي من السلوك معبي العدلة وظيفة، والتي يمكن بسهولة أن توسعت لفحص الخلايا المناعية الأخرى في نماذج التهاب الماوس.

يتم تنفيذ فحص pIL1-عن dsRed الفئران على أساس المظهرية عن dsRed إشارة مضان التي تنتجها الكريات البيض في الدم الطرفية الخاصة بهم باستخدام التدفق الخلوي. ومن المعروف LPS تحفيز للحث على إنتاج IL-1β في خلايا الدم النخاعي بما في ذلك العدلات، وحيدات، والخلايا الجذعي...

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

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

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

التصوير حيوي داخلي من العدلات الاشعال عن طريق IL-1β يحركها المروج عن dsRed الفئران مراسل

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

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.

تتبع الزحف داخل اللمعة العدلات ، والهجرة والانجذاب الكيميائي Transendothelial في الأنسجة بواسطة الميكروسكوب فيديو Intravital

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.

Intravital تصوير الغدة الصعترية باستخدام الماوس 2 فوتون الميكروسكوب

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.

Intravital التصوير من العقدة اللمفية الفأر المأبضية

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

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

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

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.

في الوقت الحقيقي التصوير من التفاعلات صفائح الدم، العدلات غيروي على البطانة الوعائية التهاب المنشط خلال تشكيل خثرة في والفئران الحية

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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.

حل غاية intravital المجهري مخطط في الإضاءة مراكز جرمينال

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.

المدى الطويل حيوي داخلي Multiphoton المجهر التصوير من الخلايا المناعية في السليمة والمريضة الكبد عن طريق الفئران CXCR6.Gfp مراسل

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.

التصوير العدلات وحيدات في مساريقي الأوردة التي كتبها حيوي داخلي المجهري على الفئران مخدرة في الوقت الحقيقي

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.

التصور من الخلايا اللمفاوية معربا-22-IL عن طريق الفئران مراسل

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.

تصوير العدلات يبلوع والسيتوبلازم باستخدام الرقم الهيدروجيني المؤشر Ratiometric

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.

تصوير بكتريا السل في الفئران مع "مراسل إنزيم الأسفار"

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