This work was supported by the Deutsche Forschungsgemeinschaft (DU 1190/3-1).

모든 동물 실험은 독일의 동물 보호 법률 (TierSchG)을 준수 하였다. FELASA (www.felasa.eu/guidelines.php) 및 국가 동물 복지 몸 GV-SOLAS (www.gv-solas.de/index.html)에 의해 정의 된 마우스는 보관과 좋은 동물 관행에 따라 처리되었다. 프라이 부르크 대학의 동물 복지위원회뿐만 아니라 지방 자치 단체 (Regierungspräsidium 프라이 부르크)는 모든 동물 실험을 승인했다. 1. 동물 취급, 준비 및 염증의 유도 <ol><li> 16-20g의 무게 범위 6 주령의 수컷 마우스 - 4를 사용합니다. 참고 : 마우스는 이전 또는 무거운 경우가 현미경 관찰을 제한, 선박을 둘러싼 과도한 지방을 제시한다. </li><li> 수술에 앞서 모든 도구와 현미경 테이블을 소독. </li><li> 주사 20 ㎎ / ㎏ LPS (리포 폴리 사카 라이드) 복강 4 세균 inflamm을 유도하는 생활 마우스로는 현미경하기 전에 시간ATION. </li><li> 플라스틱 챔버 및 장간막 조직 가습 37 ℃에서 수조에서 0.9 % 식염수 Prewarm. </li><li> 바로 현미경 절차 전에 복강 케타민 주사 (100 ㎎ / ㎏)과 자일 라진 (5 ㎎ / ㎏)와 마우스를 마취. 대안 마취는, 예를 들면, 2 % 이소 플루 란 흡입을 사용할 수있다. 반사 자극 (회사의 압력과 발가락 또는 꼬리 핀치)에 응답의 손실에 의한 적절한 마취를 확인합니다. </li><li> 면도기를 사용하여 복부를 털을 뽑다 에탄올 70 %로 포화 거즈로 느슨하게 머리를 제거합니다. </li><li> 마취 동안 건조를 방지하기 위해 마우스의 눈에 수의사 연고를 적용합니다. </li></ol> 2. 수술 <ol><li> 70 % 에탄올을 사용하여 복부 멸균. 이 방법은 멸균 방법 아니며 실험의 마지막에 치명적일 수있다. </li><li> 중간 개복술을 수행 작은 곡선 집게와 작은 가위를 사용하여 복부 피부를 엽니 다. 에피을 확인gastrical 용기는 용기를 보호하기 위해, 원격 교육 알바의 영역에서 복막을 열고. </li><li> 습윤 티슈 유지 복강 데워진 식염수 몇 방울을 적용한다. </li><li> 형광 50 μL의 로다 민 6G 9, 10 전에 설명 된 바와 같이 역 궤도 (1 ㎎ / ㎖)을 주입하여 라벨 백혈구 및 혈소판. </li><li> 10cm의 직경을 갖는 페트리 디쉬에 ileumand 장소 그것의 외면 화하다 루프 및 37 ° C를 도포하여 습윤 티슈 유지 확인은 식염수 (0.9 %), 다른 모든 순간 데워진. </li></ol> 3. 생체 내에 현미경 <ol><li> 현미경 아래에 마우스를 위치시키고 (200)의 직경과 장간막 정맥을 가지고 - 뷰의 중심에서 300 μm의. 눈에 보이는 지방 환경과 용기를 선택합니다. </li><li> 하여 내피 세포의 자극을 피 장간막 혈관을 만지지 마십시오. 조심스럽게 회장 루프를 처리합니다. </li><li> 혈액 세포 내피 상호 작용을 시각화반전 또는 직립 현미경 및 현미경 소프트웨어를 사용하여 카메라. 녹음 혈액 마우스 당 4 개의 정맥에 1 분 동안 세포 내피 세포의 상호 작용. </li><li> 촬상 실험 종료 후 경추 탈구하여 마우스를 안락사. </li></ol> 4. 분석 <ol><li> 분석 오프라인을 수행하고 모든 매개 변수에 대한 눈을 멀게. 임의의 적합한 소프트웨어 프로그램을 사용하여 분석을 수행. </li><li> 관내 피 세포의 흐름에 집중 높은 시간 해상도 씨네 클립에서 안정하고 interindividually 필적 혈류 상태 (최대 프레임 율)을 확인한다. </li><li> 롤링 백혈구의 수를 정량화. 그 때문에 정맥을 통해 수직선을 그리고 1 분에이 선을 넘어 모든 백혈구를 계산합니다. </li><li> 하나의 백혈구가 안정적으로 내피 세포에 롤링 동안 50 ㎛의 거리를 통과하는 데 필요한 시간을 측정하여 회전 속도를 결정합니다. 이 작업을 수행하려면, 50 μm의 t의 거리에 두 개의 수직 라인을 그릴정맥 hrough. <ol><li> 대표적인 백혈구 필요한 시간 (μm의 / s)으로 50 μm의 분할함으로써 other.Calculate 한 행에서 백혈구의 속도를 얻기 위해 필요한 시간을 측정한다. </li></ol></li><li> 0.04 mm (2)의 필드에 백혈구 부착을 측정한다. 이를 위해, 정맥에 200 ㎛, 변의 길이와 사각형을 그린다. <ol><li> 이 광장 내에서 30 초 동안 눈에 보이는 운동으로 정의 된 회사 부착 백혈구, 계산합니다. </li></ol></li><li> 혈소판, 백혈구 작용을 정량화하는 하나의 백혈구 결합 혈소판의 개수를 카운트. </li></ol>

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The authors have nothing to disclose.

여기에서 우리는 준비 및 생체 내에서 쥐의 장간막 정맥의 생체 내에 현미경의 성능을 설명합니다. 이 방법은 우리에게 살아있는 유기체에 직접 백혈구가 내피 세포와 혈소판 내피 세포의 상호 작용 (11)을 관찰 할 수있는 기회를 제공합니다. 내피 세포를 염증 초기 반응으로 활성화됩니다 압연, 접착력과 윤회 (12)의 결과 백혈구 및 혈소판과 상호 작용...

이 프로토콜의 목적은 염증 조건에서 백혈구 - 내피 백혈구 및 혈소판의 상호 작용의 직접적인 관찰을 위해 쥐를 거주 장간막 정맥의 생체 내에 현미경의 간략화 된 기법을 설명하기위한 것이다. 생체 내에 현미경은 염증 백혈구 모집 및 기능을 이해하는 사소한하지만 중요하지 않은 염증성 질환 1, 2에서 생체 내에서 백혈구 - 내피 상호 작용을 연구하기 위해 개발되었다. 이 프로토콜에서 우리가 설명하는 방법은 이전에 발행 된 출판물을 기반으로 개발되었다. 발표 된 내용에 성장 혈전 3 병렬로 혈소판 통합을 시각화 유사 이전 4 표면화 장간막 정맥은 투과광 현미경으로 검사한다. 이러한 쥐 5 마우스의 간 및 쥐 (6)의 cremaster 근육으로 내 생명 현미경의 다양한 모델이 있습니다. 장간막 정맥의 생체 내에 현미경이 개발되고 여러 그룹에 의해 이전의 연구에 적용되어왔다. - / - 누구의 혈소판 세로토닌 약리학 장기 고갈 된 쥐에 연구 결과 비 신경 세로토닌의 결핍과 비교 마우스, 우리는 야생형 마우스 및 트립토판의 hydroxylase1 (Tph1) 사이의 백혈구 모집의 차이를 관찰하기 위해이 기술을 사용했다 애플리케이션 선택적 세로토닌 재 흡수 억제제 인 플루옥세틴 ​​7. 우리는 또한 급성 플루옥세틴 ​​치료 (8) 후 백혈구 - 내피 상호 작용을 조사 하였다. 이 모델은 신속하게 수행하고, 백혈구 - 내피 백혈구 및 혈소판의 상호 작용의 유효 측정을 허용 할 수 있기 때문 프로토콜에서는, 장간막 정맥의 생체 내에 초점 현미경. 이것은 훨씬 더 어렵고 시간이 소요되는 등의 뼈, 간, 피부, 근육 등 cremaster 또는 다른 장기의 생체 내에 현미경이다. 모드여기에 설명 된 L은 리포 폴리 사카 라이드의 복강 내 주사로 염증 자극, 그것을 challengeing 후 염증 세포 - 세포 상호 작용의 재현성 평가에 이상적입니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;989-38-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli 055:B5</td>
			<td>Sigma-Aldrich</td>
			<td>L2637&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

intravital microscopy of leukocyte-endothelial and platelet-leukocyte interactions in mesenterial veins in mice

생체 내에 현미경 살아있는 동물에서 다른 상이한 영역에서의 프로세스와 혈관을 조사하는데 사용될 수있는 방법이다. 이 프로토콜에서는, 우리는 장간막 정맥의 생체 내에 현미경을 설명합니다. 이는 생체 내에서 내피 백혈구 작용을 도시 재현 결과 단시간에 행할 수있다. 우리는 내피 세포의 LPS 도전 후 염증 설정을 설명합니다. 그러나이 모델에 하나, 세균 화학 또는 생물학적 같은 염증성 질환의 많은 다른 유형을 적용하고 약물의 관리 및 살아있는 동물에 미치는 직접 효과와 백혈구 채용에 미치는 영향을 조사 할 수 있습니다. 이 프로토콜은 마우스 및 혈관 염증 반응에 미치는 영향의 다른 치료의 숫자에 성공적으로 적용되고있다. 여기서, 우리는 형광 로다 민 6G 이러한 레이블에 의해 백혈구 및 혈소판의 시각화를 설명합니다. 또한, 특정 영상은 PE 될 수 있습니다대상 형광 표지 된 분자를 사용하여 rformed.

이 프로토콜에 설명 된 실험 환경의 개요가도 1에 도시되어있다. 이것은 생체 내에 현미경에서 관찰 할 수 표면화 회장 루프 및 그 용기와 마우스를 보여준다.도 2 인해 미처리 동물에서 활성화의 어느 정도를 나타낸다 절차 자체. 그러나 거의 느린 압연 또는 LPS 처리 쥐에 비해 백혈구의 회사 접착이있다. 또한 야생형 마우스 중 마시는 물이나 치료 쥐 3 주 동안 플루옥세?...

Watch this Scientific Journal Video about Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice at JoVE.com

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

마우스의 Mesenterial 혈관에서의 생체 내에 현미경 백혈구 - 내피 세포와 혈소판 - 백혈구 상호 작용

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

intravital-microscopy-leukocyte-endothelial-platelet-leukocyte

Research

JoVE Journal

Immunology and Infection

12.0K 조회수.  Heart Center, University of Freiburg. This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

비디오: Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

사타구니 림프 노드의 Intravital 현미경

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

연구

면역학 및 감염병학

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.

추적 호중구 Intraluminal 크롤링, Intravital 비디오 현미경에 의한 조직의 Transendothelial 마이 그 레이션 및 Chemotaxis

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.

2 - 광자 현미경을 사용하여 마우스 Thymus의 Intravital 영상

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

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

비장의 Intravital 현미경 : 기생충 이동성 및 혈류의 정량 분석

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 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.

어린 싹 센터의 높은 해결의 intravital 스트라이프 - 조명 현미경

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.

CXCR6.Gfp 기자 마우스를 사용하여 건강하고 병에 걸린 간에서 면역 세포의 장기 생체 내에 다중 광자 현미경 이미징

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.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

똑바로 Intravital 현미경 Murine Submandibular 침 샘의 준비

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

영상 셀 상호 작용 Tracheal 점 막에서 2 광자 Intravital 현미경 검사 법을 사용 하 여 인플루엔자 바이러스 감염 중

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

병원 균, 공생, 그리고 호스트 창 자 상피 사이 상호 작용을 공부 하는 인간 Colonoid Monolayers

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...