The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

이 비디오를 게시 및 프로토콜에서 모든 동물의 절차는 분할 한 기관 바젤 슈타 트, 스위스의 승인을 동물의 프로토콜에 따라 실시 하였다. 1 림프절 준비 및 소화 <ol><li> 가열 플레이트 자석 교반기 37 ° C에 비커 초기 가열 물. </li><li> 다음과 같은 기본 미디어를 준비 : DMEM 배지 (피루 베이트 제외) 2 % FCS로 보충, 1.2 mM의 염화칼슘 및 펜 / 연쇄상 구균 (페니실린의 100 단위, 스트렙토 마이신 100 μg). </li><li> 사용하기 전에 모든 해부 도구를 소독. </li><li> CO 2 질식 당 림프절 공여 쥐를 안락사 및 무균 적으로 림프절을 해부하다. 주위의 지방을 해부하지 마십시오. 참고 :이 프로토콜은 주변 피부 배수 림프절 (사타구니, 상완, 겨드랑이)에 최적화되어 있습니다. </li><li> 2 ㎖의 차가운 얼음 기본적인 매체를 포함하는 멸균 페트리 접시에 림프절을 놓습니다. </li><li> 림프절 캘리포니아를 중단1 ML의 주사기에 고정이 25 G 바늘을 사용하여 psule. </li><li> 1 밀리그램 / ㎖의 콜라게나 제 IV 및 40 μg / ml의 DNase의 I. 보충 된 750 ㎕의 배지를 함유 염기성 5 ㎖ 폴리 프로필렌 둥근 바닥 튜브에 방해 림프절 조직을 이동시켜 </li><li> 각 튜브에서 하나의 멸균 자석 교반기를 추가합니다. </li><li> 37 ° C로 비커에 넣어 튜브는 물을 예열하고 30 분 동안 느린 속도 (1 라운드 / 초)에서 튜브를 저어. </li><li> 가열 플레이트 자석 교반기에서 튜브를 제거하고 림프절 조각이 정착 할 수 있습니다. </li><li> 조심스럽게 &quot;비 기질 세포&quot;풍부한 뜨는을 제거합니다. 참고 : T, B, 수지상 세포와 CD45의 분석 경우 - gp38 - CD31가 - 예상된다 &quot;비 기질 세포&quot;부분을 저장합니다. </li><li> 워시는 750 ㎕를 기본 매체로 한번 림프절 조직을 나머지. 참고 :이 단계는 면역 능력 마우스로 작업하는 경우에만 필요합니다. </li><li> 림프절 조각이 정착하자. </li><li> 제거비 기질 세포 부동 분수. </li><li> 750 ㎕의 기본 매체가 3.5 mg을 보충 림프절 조각에 추가 / ㎖ 콜라게나 D 40 μg / ml의 DNase의 I. </li><li> 다시 37 ° C가 들어있는 비커에 넣어 튜브는 물을 예열. </li><li> 천천히 교반하면서 5 분 동안 림프절 조직 다이제스트. </li><li> 피펫 및 자동화 된 멀티 채널 피펫을 사용하여 최대한의 속도로 10 회 700 μl를 혼합하여 해리하는 림프절 조직 조각. NOTE :이 소화를 향상시키기 림프절 조직을 방해. </li><li> 37 ° C 예열 된 물을 다시 비커에 튜브를 놓습니다. </li><li> 천천히 교반하면서 또 10 분간 다이제스트 림프절 조직 단편. </li><li> 피펫에 의해 림프절 조직 조각을 해리하는 자동화 된 멀티 채널 피펫을 사용하여 최대한의 속도로 99 사이클 혼합. </li><li> 단일 세포 현탁액의 유지 보수를 보장하기 위해 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 7.5 μl를 추가합니다. </li><li> 파이프에 의해 해리하는 림프절 조직 절편tting 및 자동화 된 멀티 채널 피펫을 사용하여 최대한의 속도로 99 사이클 혼합. </li><li> 750 ㎕의 기본 매체를 추가하고 70 μm의 나일론 메쉬를 통해 세포를 전달합니다. </li><li> 원심 분리기 세포 현탁액 1500 XG에 5 분, 4 ° C에서. </li><li> 간질 세포를 추가 분석을 위해 이제 사용될 수있다. </li></ol> 림프절 기질 세포의 2 염색 <ol><li> 성공적 TRC, LEC, BEC 및 DN 세포를 인식하는 항-CD45, 항 podoplanin (gp38), 항-CD31 항체의 조합을 사용한다. </li><li> , 안티 gp38-PE (1 : 200), 항 CD31-APC (1 : 200) 100 ㎕를 HBSS의 2를 포함 : 라이브 / 죽은 추적기, 항 CD45-FITC (200 일)로 소화 림프절 조직을 품어 어둠 속에서 20 분 이상, 4 ° C에 대한 % FCS. </li><li> 2 % FCS를 포함 HBSS의 5백리터를 추가 세포를 씻으 </li><li> 원심 분리기 세포 현탁액 1500 XG에 3 분, 4 ° C에서. </li><li> 2 % FCS를 포함 HBSS 100 ㎕에 다시 일시 중지합니다. </li><li> equipp 플로우 사이토에 염색 된 세포를 실행다음 광학 에드 : 여기까지 세 레이저를 소스 : 청색 (488 nm의, 공기 냉각, 20 mW의 고체 상태), 적색 (파장 633 nm, 17 mW의 헬륨 네온), 바이올렛 (405 나노 미터, 30 mW의 고체 상태) . </li><li> 게이트 CD45 - 세포는 조혈 세포를 제외합니다. </li><li> 게이트 단봉 (FSC-W) 및 살아있는 세포 (LIVE / DEAD 추적기) 이중선과 죽은 세포를 제외합니다. </li><li> , LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) 및 DN (gp38 - CD31 -) 세포 (그림 1 참조) - CD31 대 플롯 gp38는 TRC (gp38 + CD31)를 시각화합니다. </li></ol>

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The authors declare they have no competing financial interests.

림프절 기질 세포의 연구는 최근에 의한 소화 작용이 발행 프로토콜 6,13의 개발에 연구 초점이되었다. 두 프로토콜은 단일 림프절 간질 세포를 획득하기에 적절하지만, 소화 효소의 이용과 소화의 시간 차이가있다. 기질 세포와 그 표면 마커 효소 소화와 기계적 스트레스에 민감하기 때문에, 최적화 된 프로토콜이 필요합니다. 갓 해부 림프절에서 가능한 림프절 기?...

림프절은 외국과 자기 항원에 대한 적응 면역 반응이 시작되고 조정되는 전문 구획입니다. 여기에 제시된 절차 림프절 단일 세포 현탁액을 수득하고 여러 분자의 표면 발현을 유지 가능한 림프절 기질 세포에 접근하기 위해 자동화 된 기계적인 피펫 결합 단편의 효소 소화를 설명한다. 림프절 기질 세포가 세 가지 주요 기능을 림프 노드의 골격을 형성하고 이행 : 첫째가 체액 항원, 병원균과 병원균 관련 분자 패턴 (PAMPS)뿐만 아니라, 사이토 카인 및 위험 관련 분자 패턴을 샘플링 필터 (감쇠) 본 몸에. 둘째, 상호 작용 및 적응 면역 반응을 시작하는 항원 제시 세포 (APC)와 림프구를 유치하고 지시; 셋째, 이들은 하나의 림프구 항상성 및 분화 구조 환경을 제공-3입니다. 염증 림프절 기질 세포는 성장 인자, 사이토 카인 및 케모카인을 생산하는 동안, 수지상 세포 간의 상호 작용을 편성함으로써, T- 및 B- 세포 종기에 적응. 면역 반응의 오케스트레이션으로 인해 서로 다른 기질 세포 집단에 의해 형성되는 복잡한 구조 아키텍처에만 가능합니다. 림프절 간질 세포가 CD45 음성 세포와 섬유 모세포하며 내피 세포 1-6에서 CD31의 발현 또는 gp38에 의해 구별 될 수있다. CD31 + 혈액 내피 세포를 정의 (BEC)를 - gp38 + CD31 + 림프 내피 세포 (LEC), gp38를 정의 - Gp38 + CD31는 (섬유 모세포 망상 세포 또한 FRC로 알려진 TRC) T 존 망상 세포를 정의합니다. 또한, 특정 집단의 특성은 다른 림프절 기질 세포의 존재를 한 것으로 밝혀졌습니다. 실제로 작은 주연 세포와 같은 세포 집단은 내에 특성화시켰다gp38 - CD31 - 인구 7. 따라서, 분리 절차의 적응은 다른 림프절 기질 세포의 기능적 특성의 확인 및 특성화를 위해 유리하다. 림프절 기질 세포 소화 개발 전에 림프절 기질 세포의 연구는 조직 절편 및 현미경을 이용한 관찰에 시츄로 제한 프로토콜. 그럼에도 불구하고, 구조적 및 기능적 연구는 림프절 기질 세포의 중요한 특성을 보였다. 림프절 기질 세포는 높은 내피로 림프절의 낭하 부비동에서 림프와 연관된 낮은 분자량 단백질을 수송 도관 시스템이라고 복잡한 3 차원 구조를 형성하기 위해 podoplanin, 콜라겐과 세포 외 기질 (ECM) 단백질과 연결된 T 세포 영역에서 8 세정맥. 수지상 세포는 간질에 밀착되고 관형 콘으로 돌출 관찰 될 수있다duit 구조는 유체 샘플 8 및 항원을 검출한다. 수지상 세포와 림프절 기질 세포 (TRC를 사용해 및 수정체 상피)의 상호 작용은 케모카인의 CCL21와 CCL19 9, 10의 출시와 프리젠 테이션에 의해 매개된다. CCL19와 CCL21은 림프절의 T 세포 영역 4,11로 이전 수지상 및 T 세포를 촉진 CCR7 수용체에 의해 인식됩니다. 비슷한 케모카인을 사용에도 불구하고, 수지상와 T 세포는 림프절 (12)에 서로 다른 이동 경로를 가지고있다. 나중에, 림프절 및 순수한 림프절 기질 세포의 분리의 소화 효소를 사용하여 기능적 연구는 상이한 림프절 간질 세포의 역할과 수지상 세포와 T / B 세포 6,13와 상호 작용하는 그들의 능력에 대해 수행 하였다. 우선, IFN-γ 생산 효과기 T 세포 및 림프절 기질 세포 사이의 크로스 토크는 보조 림프 기관 14-16에서 T 세포 반응과 확산을 저해하는 것으로 대사 산화 질소의 생산을 유도한다. 둘째, 림프 N송시 기질 세포는 IL-10 (17)의 생산을 통해 규제 DC 서브 세트들의 분화를 지원하기 위해, 및 IL-7의 제조 6,18 통해 나이브 T 세포의 항상성을 조절하는 것으로보고되었다. 셋째, 림프절 기질 세포의 TLR 발현은 간질 세포가 조직 손상 중에 방출 감염 또는자가 유래의 분자 신호에 민감한 것을 시사한다. 실제로, TLR3 폴리 리간드와 림프절 기질 세포의 치료 (I는 : C)의 결과, 주요 조직 적합성 복합체 클래스 I 발현 및 공동 억제 분자 PD-L1의 상향 조절의 완만 한 상향 조절을 유도하고 있지만 보조 자극 분자 주변 조직의 극적인 변화는 식 (19) 항원. 몇몇 그룹은 림프절 기질 세포가 말초 조직 항원을 발현하고 자기 반응성 T 세포의 19,21-27 내성을 유도 보였다. 따라서, 림프절 기질 세포 및 다른 이동성 재 사이의 상호 작용을 이해sident 림프절 세포는 염증 동안에 활성 또는 면역 반응의 억제를 허용하는 새로운 표적 분자를 찾는 데 도움이 될 것이다. 따라서, 림프절의 발행 효소 분리의 구현이 필요하다. 이전에 게시 된 프로토콜은 낮은 기계적 스트레스 6,19,20와 콜라겐 기반의 효소 소화의 다른 조합을 사용합니다. 그러나 소화 효소 나 소화 효소의 상이한 조합을 가진 긴 인큐베이션 활성화 상태를 분석하고 새로운 림프절 기질 세포를 식별하기 위해 필요한 다양한 표면 분자를 저하 될. 간질 세포 분석의 유형에 따라, 링크 프로토콜 또는 프로토콜 플레쳐 더 적합 할 수도있다. 설명 된 절차에서 다소 짧은 효소 소화가 가능한 림프절 기질 세포 표면 마커의 열화를 최소화하기 위해 자동화 된 기계식 세분화와 결합된다. 이 절차는 재현성이 높은 절연을 가능하게하고낮은 변동성과 95 % 이상의 생존으로 림프절 간질 세포 집단의 차이. 갓 절연 림프절 기질 세포는 시험 관내에서 직접 작용 분석을 수행하는 기질 세포 라인의 표면 마커의 발현, 단백질 분석, 전사 연구뿐만 아니라 설정에 사용될 수있다.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
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</table>

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.

본 프로토콜로 인해 자동화 된 멀티 채널 피펫 기계 세분화에 짧은 소화 시간 (45 분 최대)로 링크 등., 2007 (6)에 의해 발표 된 수정 소화 프로토콜입니다. 또한, 상기 절차는, 더 표준화 상이한 림프절 기질 세포에 표면 마커의 열화를 최소화하고 동시에 하나 이상의 샘플의 처리를 허용한다. 의 콜라겐 IV와 콜라게나 D는 플레처 프로토콜 13에서 링크 프?...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Research

JoVE Journal

Immunology and Infection

31.0K 조회수.  University of Basel and University Hospital Basel. 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. 

비디오: Isolation of Murine Lymph Node Stromal Cells

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 ...

Murine Superficial Lymph Node Surgery

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 followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

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 ...

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 ...

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 ...

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, ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

인간과 마우스 심근 표본에서 대식세포 하위 세트 및 기질 세포의 분리

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.