이 연구는 AW, AHH에 젊은 연구자 (GIF 영)에 대한 독일 이스라엘 재단과 박사 I에 이스라엘 과학 AHH로 재단 (제 11분의 1,418) 및 (제 12분의 1,933)에서 교부금에 의해 지원되었다 . AHH 및 AW에 치과 의학의 히브리어 대학 Hadassah 대학에서 Cabakoff 연구 기금 기금.

사전에 준비 : <ul><li> PBS + 2 % FCS </li><li> 콜라게나 유형 II와 DNase의 유형 I의 1 MG / ML (샘플 당 1 ML) 2 ㎎ / ml의 PBS + 2 % FCS </li><li> 멸균 수술기구 </li><li> 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) 솔루션 </li></ul> 1. 위의 잇몸 절제 기법 <ol><li> 승인 IACUC 지침을 사용하여 쥐를 안락사. </li><li> 뺨과 날카로운 / 무딘 똑바로 가위로 하악 무스 등 구강의 양쪽을 잘라. </li><li> 하악을 아래로 당깁니다. </li><li> 표준 가위 분의 어금니 뒤에 가상의 선 1mm의 소프트 및 하드 조직을 잘라. 가위 구개 뼈 (그림 2A, 파란색 선)의 평면에 수직 연출합니다. </li><li> 소프트 및 하드 조직에게 앞니 (그림 2A, 파란색 선) 뒤에 2mm를 절개. </li><li> 입의 앞쪽 부분을 아래로 당깁니다. 비강이 단계 이후에 관찰된다. </li><li> 가위를 배치구개에 평행, 비강으로 한 잎을 넣어, 다른 날은 현관에서 절개한다. 후방 절개 (그림 2A, 녹색 선)까지 잘라냅니다. 상악의 양쪽에서이 단계를 반복합니다. 끝에서 상악은 두개골의 나머지 부분 (그림 2B)에서 분리된다. </li><li> , 상악를 제거 중간 봉합 (그림 2, 검은 점선)에 잘라 치조골에 도달 할 때까지 각 헤미 - 상악의 구개 조직을 잘라. </li><li> Adson 집게 (치아 제외)과의 앞쪽에 국경에서 치은 조직 (두 헤미 - 상악) 껍질. </li><li> PBS + 2 % FCS와 함께 접시에 절제 잇몸를 놓습니다. </li></ol> 2. 잇몸 처리 <ol><li> 1 ML PBS와 함께 조직 배양 접시 (35 × 10 ㎜)에 절개 잇몸을 넣고 + 2 % FCS, 콜라게나 유형 II와 DNase의 유형 I. 1 MG / ML 2 MG / ML </li><li> N ° 15 불임 수술 블레이드와 잘 조직을 말하다. </li><li> TR접시에서 15 ML 원뿔 시험관 조직을 ansfer. </li><li> 1 ML PBS + 2 % FCS와 동일한 테스트 튜브로 전송과 함께 접시에 남아있는 조직 / 세포를 씻으십시오. </li><li> 37 20 분 셰이커 인큐베이터에 품어 ° C, 200 rpm으로 (추천 : 예열 인큐베이터). </li><li> 37 또 다른 10 분 ° C, 200 rpm으로 20 개의 EDTA (에틸렌 다이아 민 테트라 초산) 0.5 M의 μL와 부화를 추가합니다. </li><li> 12 PBS + 2 % FCS와 ML 4에서 원심 분리기까지 테스트 튜브를 채울 ° C, 8 분 400 XG (추천 : 사전 냉기 원심 분리기). </li><li> 2 ML PBS + 2 % FCS로 뜨는 세포를 resuspend를 제거합니다. </li><li> 70 μm의 셀 스트레이너와 샘플을 필터링을 통해 흐름을 저장합니다. </li><li> 4 ° C, 5 분 320 XG에 원심 분리기. </li><li> 300 μL PBS + 2 % FCS로 뜨는 세포를 resuspend를 제거합니다. </li><li> 세포를 (약 5-10 × 10 5 세포는 단일 상악에서 예상되는) 계산. </li></ol> 3. 세포와 세포 내 항체유동 세포 계측법 분석을위한 염색 그것은 소등와 후드 내부에서 작업 할 것을 권장합니다. 그것은 지속적인 추운 환경에서 세포를 유지뿐만 아니라 필요한 모든 재료하는 것이 중요합니다. <ol><li> 100 μL의 총 부피 1 X 10 6 세포 당 선택한 각 항체의 0.2-0.5 μg을 추가하여 세포 분자에 세포를 얼룩. </li><li> 소용돌이 짧게. </li><li> 4 ° C.에 어둠 속에서 15 분 동안 튜브를 품어 </li><li> 2 ML PBS + 2 % FCS와 세포를 씻으십시오. </li><li> 4 ° C, 5 분 320 XG에 원심 분리기. </li><li> 추가 뜨는 세포를 resuspend를 대기음, 현명한 드롭, 500 μL 차가운 BD는 Cytoperm / Cytofix 소용돌이로 교반하면서. </li><li> 4 40 분 ° 어둠 속에서 C에 대한 튜브를 품어. </li><li> 소용돌이 짧게. </li><li> 1 ML 차가운 BD 파마로 두 번 세포를 씻으 / 4시 7 분 800 XG에 버퍼와 스핀을 씻으 ° C. </li><li> 300 μL를 제외하고 뜨는을 대기음. </li><li> 에 의해 세포 내에서 세포를 얼룩100 μL의 총 부피 1 X 10 6 세포 당 선택한 각 항체의 1 μg을 추가 할 수 있습니다. </li><li> 소용돌이 짧게. </li><li> 4 ° C.에 어둠 속에서 30 분 알을 품다 </li><li> 1 ML 차가운 BD 파마로 두 번 세포를 씻으 / 4시 7 분 800 XG에 버퍼와 스핀을 씻으 ° C. </li><li> 300 μL 차가운 BD 파마 / 워시 버퍼를 추가하여 상층 액과 세포를 resuspend을 대기음. </li><li> 소용돌이 짧게. </li><li> FACS 튜브 필터링을하는 동안 현탁액을 전송합니다. </li><li> 분석까지 어둡고 추운 환경에서 튜브를 유지합니다. </li></ol>

<ol>
	<li>Arizon, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss.">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li><li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

저자는 공개 아무것도 없어.

하나의 마우스에서 얻은 상악 치은 조직은 우리가 이전 1 설명한대로 세포와 세포 내 분자를 표현하는 T 및 B 림프구의 하위 인구뿐만 아니라, 자신의 능력을 분석하기에 충분합니다. 그럼에도 불구하고, 희소 한 세포 집단 (예를 수지상 세포에 대한) 관심이 있으며, 그것은 2-3 생쥐 수영장 조직에 권장됩니다. 참고로, 바람직하는 경우, 그것은 할 수 있습니다 다음, 구개 치은 조직 및 소비?...

잇몸은 치아의 치경부 부분을 둘러싸고있는 연부 조직과 폐포 과정 (그림 1)을 다룹니다. 치은 더 점막 상피 세포와 결합 조직 (또한 점막하 또는 고유 층라고도 함)로 분리 할 수​​ 있습니다 저작 점막의 유형입니다. 치은 및 인접 치아의 해부학 적 구조는 박테리아가 지속적으로 로컬 면역 체계를 도전 플라크 (바이오 필름)을 상주하고 발전 할 수 있습니다. 치주 질환 2를 칭했다 조건 - 결과적으로 염증 반응은 특정 상황에서 파괴되고 잇몸에 개발하고 있습니다. 기본적으로, 플라크에 의한 치주 병변은 치은염과 치주염으로 나눌 수 있습니다. 치은염은 잇몸에 국한되는 로컬 염증 반응의 가역적 인 상태를 나타냅니다. 치주염은, 다른 한편으로는, 돌이킬 수없는 파괴적인 과정이다하는 부착 장치 (치조골, 치주인대, 백악질과 치은은 3) 파괴된다. 잇몸이 치주 질환 4시 등의 이펙터와 유도 두 사이트를 제공하기 위해 제안되었다. 인간의 연구는 치과 플라크에 대한 응답으로, 면역 효과기 세포와 분자가 동적으로 침투하거나 출발 잇몸 5-7 것을 제안했습니다. 이 활동은 치주 파괴 8,9에 중요한 역할을하는 것으로 나타났다. 인간의 조직 작업이 병적 과정에 대한 귀중한 정보를 제공하는 연구에 의해 생성 된 데이터는 중요한, 윤리적 기술 및 실험 한계를 가지고 반면. 형질 전환 생쥐를 이용한 통해 생체 개입 10 인과 실험을 허용 실험 모델의 개발. 그 결과로, 치주 질환에 관여하는 메커니즘에 대한 우리의 지식은 지난 20 년간 크게 증가했다. 그럼에도 불구하고, 치주 질환의 복잡성으로 인해,이조직의 파괴를 촉진 지역 면역 반응의 특성에 관한 지속적인 논의. 치주 질환시 치은 중앙 면역 세포의 기능에 대한 이해의 부족이있다. 그것은 질병, 잇몸의 표적 조직에서 발생하는 병적 인 염증 이벤트를 공부 때문에 필수적이다.

<table border="1">
 <tbody>
 <tr>
 <td>Comments (optional)</td>
 <td>Catalogue number</td>
 <td>Company</td>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td></td>
 <td>CLS-2</td>
 <td>Worthington Biochemical Corp.</td>
 <td>Collagenase Type II</td>
 </tr>
 <tr>
 <td></td>
 <td>DN25-1G</td>
 <td>SIGMA</td>
 <td>DNAse I</td>
 </tr>
 <tr>
 <td></td>
 <td>E6758-100G</td>
 <td>SIGMA</td>
 <td>EDTA</td>
 </tr>
 <tr>
 <td></td>
 <td>D8537</td>
 <td>SIGMA</td>
 <td>Dulbecco's PBS</td>
 </tr>
 <tr>
 <td>Heat Inactivated</td>
 <td>04-121-1</td>
 <td>Biological Industries</td>
 <td>Fetal Bovine Serum </td>
 </tr>
 <tr>
 <td></td>
 <td>FPE-204-500</td>
 <td>Jet Biofil</td>
 <td>Vacuum-Driven Filter</td>
 </tr>
 <tr>
 <td></td>
 <td>352052</td>
 <td>BD Falcon</td>
 <td>5 ml Polystyrene Round-Bottom Tube</td>
 </tr>
 <tr>
 <td></td>
 <td>93070</td>
 <td>SPL Lifesciences</td>
 <td>Cell Strainer 70 &mu;m</td>
 </tr>
 <tr>
 <td></td>
 <td>153066</td>
 <td>NUNC</td>
 <td>Tissue Culture Dish 35&times;10 mm</td>
 </tr>
 <tr>
 <td></td>
 <td>554714</td>
 <td>BD</td>
 <td>BD Cytofix/Cytoperm</td>
 </tr>
 <tr>
 <td>Clone N418</td>
 <td>117305</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD11c antibody</td>
 </tr>
 <tr>
 <td>Clone 104</td>
 <td>109819</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD45.2 antibody</td>
 </tr>
 <tr>
 <td>Clone GK1.5</td>
 <td>100413</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD4 antibody</td>
 </tr>
 <tr>
 <td>Clone 53-6.7</td>
 <td>100733</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD8a antibody</td>
 </tr>
 <tr>
 <td>Clone 17A2</td>
 <td>100214</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD3 antibody</td>
 </tr>
 <tr>
 <td>Clone G8.8</td>
 <td>118219</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD326 (Ep-CAM) antibody</td>
 </tr>
 <tr>
 <td>Clone 929F3.01</td>
 <td>DDX0362D</td>
 <td>Imgenex</td>
 <td>Anti-mouse CD207 (Langerin) antibody</td>
 </tr>
 <tr>
 <td>Clone 39-10-8</td>
 <td>115010</td>
 <td>Biolegend</td>
 <td>Anti-mouse I-Ad (MHC-II) antobody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>BD Biosciences</td>
 <td>LSR II Flow Cytometer</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>Tree Star</td>
 <td>FlowJo Software v 7.6.5</td>
 </tr>
 </tbody>
</table>

isolation, processing and analysis of murine gingival cells

우리는 정확하게 분리 및 유동 세포 계측법 및 분자 연구를위한 쥐 치은 조직을 처리하는 기술을 개발했습니다. 치은이 치주 질환을 일으킬 수 있습니다 구강 바이오 필름에 대한 숙주 면역 반응에 관여하기 때문에 면역 메커니즘을 연구하는 독특하고 중요한 조직이다. 또한 치조골 조직에 치은의 근접도 염증 상태에서 뼈의 리모델링을 공부 가능하게합니다. 우리의 방법은 우리가 이전에 1을 보여 같은 랑게르한스 세포와 T 조절 세포로도 희귀 한 세포 집단의 분석을 허용 면역 세포의 다량을 얻을 수 있습니다. 치주 질환시 치조골의 손실에 관련된 지역의 면역 반응을 연구하는 고용 쥐 때문에 다양한 면역 실험 도구의 가용성 유리합니다. 그럼에도 불구하고, 그들의 작은 크기와 쥐 잇몸에 상대적으로 불편 접근으로 인해 많은 연구가 생의 검사를 피할 수의 중요한 조직. 이 작품에서 설명하는 방법은 희망 치주 질환시 경구 면역 시스템과 그 역할을 우리 과소을 증가 잇몸 분석을 용이하게 할 수 있습니다.

잇몸 세포 유동 세포 계측법 분석의 예를 제시한다. 2 마우스에서 풀링 된 치은 세포 LSR II 흐름 cytometer에에서 실행 FlowJo 소프트웨어를 사용하여 분석 하였다. 그림 3A는 사이드 분산 형 (SSC) 대 앞으로 분산 형 (FSC) 플롯 순진 생쥐에서 치은 세포의 분포를 보여 주었다. 식별 할 수 게이팅 전략 (I) 림프구 (II) 단핵구 / 돌기 세포 그리고 (iii) 과립구가 표시됩니다. 비교 목적을 위해, 우리?...

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Isolation, Processing and Analysis of Murine Gingival Cells

이 연구는 단일 셀 문화를 생산하기 위해 마우스 구강의 잇몸 조직을 분리하고 처리 할 수​​있는 효율적인 방법을 설명합니다. 그 결과 세포는 더욱 유동 세포 계측법 분석 및 분자 연구에 사용할 수 있습니다.

쥐과 잇몸 세포의 분리, 처리 및 분석

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

isolation-processing-and-analysis-of-murine-gingival-cells

Research

JoVE Journal

Immunology and Infection

18.7K 조회수.  Hebrew University - Hadassah Medical Center. 이 연구는 단일 셀 문화를 생산하기 위해 마우스 구강의 잇몸 조직을 분리하고 처리 할 수​​있는 효율적인 방법을 설명합니다. 그 결과 세포는 더욱 유동 세포 계측법 분석 및 분자 연구에 사용할 수 있습니다.

비디오: Isolation, Processing and Analysis of Murine Gingival Cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Murine Aortas 이내에 면역 세포의 흐름 Cytometry 분석

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

연구

면역학 및 감염병학

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

격리, 식별 및 쥐과 흉선 상피 세포의 정화

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

쥐 복막 대 식세포의 분리는 수신자 같은 수용체 자극하면 유전자 발현 분석을 수행하기

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

분리 및 쥐 림프구의 활성화

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

분석 Murine 가장자리 지역 B 세포 생체 외에서의 전단 흐름 유도 마이그레이션

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...