우리는 Sylvie의 Lesage 및 프로토콜의 중요한 독서에 대한 모든 실험실 구성원 감사드립니다. 이 작품은 건강 연구 (CIHR)의 캐나다 연구소에서 교부금 (걸레-77545)에 의해 지원되었다. 나탈리 Labrecque는 FRSQ 수석 장학금의 지원과 멜리사 매튜는 NSERC 알렉산더 그레이엄 벨 캐나다의 대학원 장학금을 받고있다.

1. 수술 전 준비 마우스는 동물 복지 지침에 캐나다 문화원에 따라 취급되었다. <ol><li> 케타민을 / xylazine (300분의 150 밀리그램 / kg, IP)의 주입에 의해 또는 isoflurane의 흡입 (2 %, 1L 산소)에 의한 마취시키다 생쥐. 가능한 경우는 마취 시간과 깊이 나은 제어를 허용로 isoflurane를 사용해야합니다. 또한 산소는 동물의 생리 기능을 지원하는 동시에 관리합니다. 또한, isoflurane 직접 폐에 의해 제거되며 따라서 간장이나 신장 대사가 필요하지 않습니다. </li><li> 또는 다른 진통제 - buprenorphine를 (0.1 밀리그램 / kg, SC 0.05) 주사. </li><li> (isoflurane을 사용하는 경우) 안구 건조를 피하기 위해 수술 전에 마우스 눈을에 연고를 바릅니다. </li><li> 마우스가 앞발을 곤란하고 꼬리를 압연하여 자고 있는지 확인합니다. 마우스가 반응하지 않는 경우 수술로 진행합니다. </li></ol> 2. 외과 <p클래스 = &quot;jove_content&quot;&gt; 그것은 수술에 의해 외면적인 림프절 (후면)의 4 제거할 수 있습니다 :이 사타구니와 2 상완. 후면의 격리 에선 절연을 방해할 수있는 나이가 생쥐의 지방 조직의 존재 이후 젊은 린 생쥐 (6~8주)에 더 쉽습니다. 그러나,이 제한은 반복 연습으로 극복하고 있습니다. 쥐 편안함을 위해, 우리는 한 번에 두 LNs 최대 수확과 우리가 시간 코스 실험을 수행할 때, 우리는 매초 일 LNs을 수확. 또한, 감염을 막기 위해 항생제 마우스에 주어진, 그러나 우리는 그렇지과 상처는 잘 치료하고 수 있습니다. 실험 계획법에 따라 항생제의 사용 결과 (예 : 세균에 대한 응답을 측정하는 경우) 관찰 영향을 미칠 수 있다고 인식되고 하나는 필요합니다. 모든 악기는 각 사용 후에 소독하고 무균 조건이 절차 내내 유지하고 있습니다. <ol><li> 그 측면에 마우스를 올려. </li><li> 당신 incisio을 수행합니다 지역을 집중하다N, 당신이 적출하고 싶습니다 후면에 따라. (그림 1). </li><li> 해당 지역에, chlorhexidine, 또는 다른 방역을 적용합니다. </li><li> 날카로운 가위 (5mm 정도) foreleg의 뒷면이나 복부 근처의 뒷다리 다쳤구나 (그림 1) 이상 중 하나와 작은 절개를합니다. </li><li> 당신 포셉으로 차단 느슨한 머리카락을 제거합니다. 원하는 경우에는 절개를하기 전에 마우스를 면도 있습니다. 그러나 절차를 길게하고 우리가 여기와 같이, 그것은 모두 매우 드물게 치유하거나 감소하는 감염을 개선하지 않기 때문에 그것이 필요하지 않을 것을 발견했습니다. </li><li> 후면을 볼 수 있도록 두 집게로 절개를 부풀. 절개는 10mm에 도달할 수 있습니다. 아까 잿빛 또는 주변의 지방보다 더 어두운 나타납니다. </li><li> 한 집게로 종이 위에 근막을 (지방과 조직을 덮고있는 얇은 막) 꼬집어와 주변 조직을 파괴하지 않고 가볍게 당기십시오. </li><li> 까지 가능한 unde 번째 집게를 놓고그 종이를 rneath. 첫 포셉으로 근막을 끊어도하고 종이를 제거합니다. 당신이 성공적으로 종이를 수확 경우 혈액 얼룩이 종이는 이전에 위치해 있던 위치에 나타납니다. 참고 isotonic 솔루션 에선 싱크. 이것은 간단한 테스트 당신이 종이와 지방없는 조직을 추출한 것을 확인할 수 있습니다. </li><li> 더 머리카락이 상처에 없는지 확인합니다. </li><li> (피부의 안쪽) 함께 피부의이 부분을 고집 의해 클립으로 그들을 stapling하여 절개를 닫습니다. 우리는 적용 포셉 함께 미셸 클립을 사용합니다. 절개의 크기에 따라 1 개 또는 2 클립을 설치합니다. 조직 고치면 가장 자주 클립이 떨어져. </li></ol> 3. 수술후 관리 <ol><li> 생쥐는 수술 후 쉽게 먹을 수 있도록 새장 바닥에 약간의 서부 유럽 표준시 음식을 추가합니다. </li><li> 수술 후 6-8시간에 대하여, 그들이 고통을 경험하지 않도록하기 위해 생쥐를 확인합니다. 신중하게 그들의 행위 (자세, 걸음걸이, 관련 사항을 준수모피의 congener 및 외관과 teraction). 생쥐의 전반적인 외관이 가난한 경우 buprenorphine의 두 번째 복용을 관리할 수 있습니다. 다음날으로 쥐도 buprenorphine의 관리를 계속 달리 그들의 정상적인 동작을 회복해야합니다. 마우스는 이전 결정 및 동물 관리위원회의 승인을 종점에 도달하지 않습니다. 당신이 조언을 추구하고 동물 케어 동식물 사육장 기술자의 도움을하시기 바랍니다. </li></ol> 4. 림프절 처리 <ol><li> 실험실에서 단일 세포 현탁액을 얻기 위해, 평소 절차에 따라 그 종이를 떼어 놓다. 우리는 프로스트 슬라이드 반대 하냐구요을 누르면 일상적 에선 당 1-2 X 10 6 세포를 구하십시오. 또한, 우리는 RNA 추출을위한 종이를 준비합니다. </li><li> 평소 프로토콜에 따라 세포 청바지. 당신이 예상 하냐구요 유동세포계측법 프로필을 취득해야합니다. </li></ol> 5. 대표 결과 순방향 및 사이드 분산형 P의 예많은 수술이나 희생 마우스 (그림 2)에서 anesthetized 마우스에서 수확 LNs에 대해서만 표시됩니다. 라이브 림프구 게이트의 프로필과 세포의 비율은 솔직히 수술은 수확 세포에 적절한 방법임을 보여주는 동일합니다. <img alt="그림 1" src="/files/ftp_upload/3444/3444fig1.jpg" /> 그림 1. 사타구니와 마우스의 상완 하냐구요 지방화. A. 사타구니 에선 약간 배쪽으로 그냥 뒷다리 다쳤구나 위에 위치하고 있습니다. B. 상완 에선 foreleg 뒤에 위치하고 있습니다. <img alt="그림 2" src="/files/ftp_upload/3444/3444fig2.jpg" /> lymphocytes의 그림 2. 유동세포계측법 프로필 에선 수술에 따라 획득하였습니다. 전방 측면 분산형 프로파일 anesth에서 수확 LNs 참고해 주시길 바랍니다수술 ()에 의해 또는 희생 마우스 (B)에서 etized 마우스. LNs는 수확 dissociated 및 유동 cytometer를 분석했다. 라이브 림프구 게이트에있는 세포의 비율이 표시됩니다.

<ol>
	<li>Zehn, D., Lee, S. Y., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+but+curtailed+T-cell+response+to+very+low-affinity+antigen.">Complete but curtailed T-cell response to very low-affinity antigen.</a> Nature. 458, 211-214 (2009).</li><li>Vezys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8+T-cell+compartment+grows+in+size+with+immunological+experience.">Memory CD8 T-cell compartment grows in size with immunological experience.</a> Nature. 457, 196-199 (2009).</li><li>Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+persistence+of+memory+CD8(+)+T+cells+depend+on+T+cell+factor+1.">Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.</a> Immunity. 33, 229-240 (2010).</li><li>Wirth, T. 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관심의 어떠한 충돌 선언 없습니다.

우리는 많은 실험적인 시스템에 적용할 수있는 에선 수술의 프로토콜을 설명했습니다. 면역 반응 또는 면역 세포 항상성를 수행하는 것이 매우 유용하지만,이 기법은 몇 가지 제한 사항이 있습니다. 첫째, 적당히 LNs을 수확 할 수 있습니다. 둘째, 공부 면역 반응은 조직의 산물이거나 외면 사타구니 또는 상완 (피부 배수) LNs에서 발생합니다. 마지막으로, 수확 세포의 숫자 에선 제거, 절대 셀 참조...

<table border="1"><tbody><tr><td> 시약 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 파인 패턴 프리미엄 집게를 Graefe </td><td> 하버드 장치 </td><td> 52-2144 </td><td> 매우 구부러져, 10cm (4), 0.8 밀리미터 팁 </td></tr><tr><td> 미셸 클립 집게 적용 및 제거 </td><td> 하버드 장치 </td><td> 52-3779 </td><td> 12.5 cm (5) </td></tr><tr><td> 미셸 클립 100 </td><td> 하버드 장치 </td><td> 52-3746 </td><td> 7.5-1.75 mm </td></tr><tr><td> Temgesic (Buprenorphine 염산염) </td><td> Schering-북두칠성 </td><td></td><td></td></tr><tr><td> Vetalar (케타민을 하이드로 클로라이드) </td><td> Boniche 동물 건강 관리 </td><td></td><td> DIN : 01,989,529 </td></tr><tr><td> Rompun (Xylazine) </td><td> 바이엘 </td> &lt;TD&gt; </td><td> DIN : 02,169,592 </td></tr><tr><td> Isoflurane </td><td> 애보트 </td><td></td><td> DIN : 02,032,384 </td></tr><tr><td> Hypotears의 눈 연고 </td><td> 노바티스 Ophtalmics </td><td></td><td> DIN : 02,133,288 </td></tr><tr><td> Baxedin (이소 프로필 알코올의 Clorhexidine 글루 콘 산 2퍼센트 70 %) </td><td> 오메가 </td><td> L0000017 </td><td> DIN : 02,251,477 </td></tr></tbody></table>

murine superficial lymph node surgery

백신, 감염이나 종양에 대한 면역 반응의 진행을 이해하는 면역학 분야에서, 반응은 종종 시간이 지남에 따라합니다. 마찬가지로 림프구 항상성의 연구는 시간 과정 실험이 필요합니다. 같은 마우스 내에서 이러한 연구를 수행하는 것은 실험적 다양성뿐만 아니라 사용하는 마우스의 개수를 줄이기 위해 이상적입니다. 혈액 금단 시간 과정 실험의 성능을 가능하지만, 그것은 단지 lymphocytes 순환에 대한 정보를 제공하고 세포 1-4 제한된 수의를 제공합니다. 몸 전체 순환과 림프 노드에 거주하는 lymphocytes 서로 다른 성질을 가지고 있기 때문에 두 위치를 검토하는 것이 중요합니다. 수술에 의한 림프절의 연속적인 제거는 면역 반응이나 시간의 경과에 같은 마우스의 면역 세포 확장을 수행하는 독특한 기회를 제공합니다. 또한이 기술은 PE에 대한 충분한입니다 림프절 당 1-2x10 6 셀 사이에 듭니다phenotypic 특성화 및 / 또는 기능 assays를 rform. 순차 림프절 수술이나 lymphadenectomy이 성공적으로 우리와 다른 5-11에 의해 사용되었습니다. 여기서는 상완과 사타구니 임파선이 anesthetised 마우스의 피부에 작은 절개를함으로써 제거할 수 방법에 대해 설명합니다. 수술은 외면하고 신속하게 이루어지기 때문에, 마우스가 매우 빠르게 복구 잘 치유하고 과도한 통증을 경험하지 않습니다. 매초마다 하루 시간 과정 실험 허용 하나 또는 두 개의 림프절을 적출하는 것이 가능합니다. 이 기술은 시간이 지남에 임파선의 특성 노드 거주 lymphocytes를 공부하기 때문에 적합합니다. 이 방법은 여러 가지 실험 설계에 적합하며 우리는 많은 실험실은 순차 림프절의 수술을 혜택을 누릴 것이라고 믿습니다.

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

같은 마우스 내에서 시간에 따른 면역 반응의 진행을 수행하려면, 임파선이 순차적으로 수술로 제거할 수 있습니다. 여기서는이 기술을 수행할 수있는 방법에 대해 설명합니다.

Murine 외면적인 림프절 외과

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Research

JoVE Journal

Immunology and Infection

41.9K 조회수.  Maisonneuve-Rosemont Hospital Research Center. 같은 마우스 내에서 시간에 따른 면역 반응의 진행을 수행하려면, 임파선이 순차적으로 수술로 제거할 수 있습니다. 여기서는이 기술을 수행할 수있는 방법에 대해 설명합니다.

비디오: Murine Superficial Lymph Node Surgery

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

위드 필드와 공촛점 현미경과 Murine 림프절의 조각에서 T 세포의 생체내 이미징 전

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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.

마우스 오금 림프절의 Intravital 이미징

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

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

쥐과 림프절 기질 세포의 분리

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

약독 화와 피내 접종에 따라 마우스의 피부와 배수 림프절에서 골수 세포 분리<em&gt; 변형체</em&gt; 포자 소체

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

쥐의 전체 두께 피부 이식

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

세포 내 염색 및 유동 세포 계측법은 고혈압의 모델에 쥐 대동맥, 신장 및 림프절에서 림프구 하위 집합을 식별하는

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

수집 및 RNA 분석에 대 한 큰 동물에서 림프절의 처리: 큰 동물 종의 림프 노드 Transcriptomic 연구에 대 한 준비

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Murine Thymus, 림프절, 비장 Cytometry 사용 하 여 배출 췌 장 하위 집합 규제 T 세포의 결정

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...