이 작품은 (KAH에) 건강 그랜트 R01 AI088209 국립 연구소에 의해 지원되었다. 우리는 또한 미네소타 유동 세포 계측법 자원의 대학 감사합니다.

본 연구에서는, 성인 (6 - 8 주) 암컷 C57BL / 6 쥐를 사용 하였다. 마우스는 국립 암 연구소에서 구입 한 특정 병원균이없는 상태에서 유지되었다. 미네소타 기관 동물 관리 및 사용위원회의 대학 (IACUC)는 모든 동물 실험을 승인했다. 인스 트루먼 트와 버퍼의 1 준비 <ol><li> 효소 용액을 준비 알부민 풍부한 완충액 (PBS 1X [칼슘 / 마그네슘 2 + 불 함유]와 0.5 % 소 혈청 (0.05 %와 RPMI1640 배지를 Liberase TH의 DNase I 및 100 U / ㎖ [/ w V]) 알부민 및 2 mM의 에틸렌 디아민 테트라 산 [EDTA]), FACS 완충액 (1X PBS [칼슘 / 마그네슘 2 + 불 함유 함유하는 1 % 태아 소 혈청 [FBS] 및 0.02 % NaN3가), 버퍼 정렬 FACS (1X PBS [칼슘 / 마그네슘이 2 + 불 함유] 2 % FBS), RP10 및 배지를 함유하는 (RPMI-1640 배지)는 10 % FBS와 함께 공급된다. </li><li> 10 패닝 접시를 준비합니다. (0.01 ㎎ / ㎖ G 40 ml의 코팅 용액을 준비귀리는 0.1 M 탄산 수소 나트륨 완충액 안티 쥐의 IgG), 각 100 × 15mm 플레이트, 소용돌이로 코팅 용액 4 mL를 넣고 4 ° CO / N 부화. 항체 용액을 붓고 두 번 1X PBS 5 ㎖를 사용하여 판을 세척, 헹굼 사이에 격렬하게 소용돌이 친다. 4 ° C에서 제조 된 판을 보관하십시오. </li></ol> 흉선 조직의 2 수확 <ol><li> CO 2 챔버에 마우스를 안락사. , 해부 매트에 그 뒷면에있는 동물을 넣어 매트에 발을 핀 및 70 % 에탄올로 분사하여 모피 젖은. </li><li> 똑바로 가위로 아래턱에 요도 위에서 시작하여 피부를 통해 중간 선 절개를하고 양쪽 무릎 아래쪽으로 절개를 확장 할 수 있습니다. 절개는 &quot;Y&quot;거꾸로처럼 보인다. 측면에 느슨한 피부를 뒤로 당겨 절개 매트에 핀. </li><li> , 검상 돌기에서 진동판에 구멍 쇄골에 대해 다음 갈비뼈를 들어 올려까지 양쪽의 갈비뼈를 잘라집게 해부 매트에 고정합니다. 흉선은 갈비뼈 아래에, 그리고 마음 (그림 1) 위에 놓인 두 개의 얇은 흰색 엽 (叶)처럼 보인다. 분리 결합 조직을 부드럽게 당겨 곡선 톱니 모양의 집게와 흉선 로브의 쌍을 제거, 흉선 주변. 5 ㎖의 RPMI-1640을 포함하는 6 웰 플레이트에 배치 흉선 엽. </li></ol> 흉선 기질 세포의 3 준비 <ol><li> 모든 주위 지방 및 결합 조직을 손질하고 5 ml의 갓 효소액을 제조 함유 6 웰 플레이트의 웰에 새로운 흉선 로브 옮긴다. 미세 가위로 흉선 엽 (叶)에 작은 절개를 확인하고 20 분 동안 37 ° C에서 접시를 품어. </li><li> 조직 해리 5 ml의 피펫을 사용하여 3 회 - 부드럽게 흉선 조직 상하 2를 소화 피펫. 10 ㎖의 효소를 중화 알부민 풍부한 차가운 완충액을 함유하는 50 ㎖의 튜브에 상청 분획을 수집한다. 얼음에 포집 관을 유지합니다. 2.5 추가ML 효소 나머지 조직에 솔루션을 15 분 동안 37 ° C에서 접시를 품어. </li><li> 골재 분리를 위해 3 ML의 주사기와 18 G 바늘 부드러운 기계적 교반을 수행합니다. 포집 관에 풀 상층 액. 나머지 조직에 2.5 ml의 효소 솔루션을 추가하고 15 분 동안 37 ° C에서 접시를 품어. </li><li> 3 ML의 주사기와 25 G 바늘 부드러운 기계적 교반을 반복 한 접시 추가 5 품어 - 10 분. </li><li> 완전한 소화 한 후, 포집 관에 뜨는을 풀. 원심 분리기 400 XG, 8 분 세포를 수집하기위한 4 ° C에서 포집 관. 대기음 액체 및 10 ㎖ 알부민 풍부한 버퍼에 펠렛 세포를 중단하고 100mm 메쉬를 통해 샘플을 필터링. 혈구를 사용하여 세포를 계산합니다. 그런 흐름 cytometic 분석 (제 4 절) 또는 TEC는 농축 (5 장)을 수행합니다. </li></ol> 유동 세포 계측법에 의해 TEC는의 4 확인 </p&gt; <ol><li> 96 웰 둥근 바닥 플레이트에서 테스트에 대한 FACS 염색 버퍼 100 μL 당 4 × 106 세포의 최종 농도로 세포를 준비하고 얼음에 플레이트를 유지한다. 세포 (10 μg FACS 버퍼 / ㎖에서 항 CD16 / CD32 항체) 20 μL의 Fc 블록 솔루션을 추가하고 얼음에 10 분 동안 세포를 배양한다. 400 XG, 3 분 동안 4 ° C에서 접시를 스핀. 싱크대 아래로 판의 얼굴을 쓸어 넘겨 우물에서 액체를 폐기하십시오. </li><li> 펠렛 세포 100 ㎕의 항체 칵테일 (항-CD45, -EpCAM, -MHC 클래스 II, 및 -Ly51 항체, 및 제조 업체에서 서로 다른 형광 색소로 표지 UEA-1 렉틴은 권장 또는 FACS 버퍼에 각 항체의 최적 농도를 시험 일시 중단 ) 및 어둠 속에서 20 분 동안 얼음 위에서 세포를 배양. 보상의 경우, 각각의 형광 색소와 1 × 10 6 세포를 염색. </li><li> 각 웰에 FACS 버퍼의 100 μl를 추가하고 400 XG, 3 분 동안 4 ° C에서 접시를 회전. 세척 단계를 반복시간 후 100 ㎕의 FACS 완충액에 세포를 재현 탁. 300 ㎕의 FACS 버퍼 가득 12 × 15mm 둥근 바닥 FACS 튜브에 세포를 전송합니다. 유동 세포 계측기에 샘플을 획득하고 FACS 분석 소프트웨어를 사용하여 데이터를 분석한다. TEC 게이팅 전략은 그림 2에 나타내었다. </li></ol> 패닝에 의해 TEC는의 5 심화 <ol><li> 400 XG, 5 분 동안 4 ° C에서 (제 3에서 제조) 흉선 세포를 스핀 다운. 대기음 액체 및 15 ㎖의 50 ml의 원추형 튜브 ML 당 2 × 108 세포의 농도로 FACS 정렬 버퍼에 펠렛 세포를 일시. 세포 현탁액의 2.5 ㎍ / ml의 최종 농도 방지 CD90.2 항체 (클론 30-H12) (또는 항 CD45)을 추가하고 30 분 동안 4 ° C에서 회전 믹서에 부드럽게 세포를 배양한다. 배양 후, 5 씻어 - RP10 매체의 10 배 볼륨과 400 XG, 5 분 동안 4 ° C에서 튜브를 원심 분리기. </li><li> 대기음 액체의 펠렛 세포를 재현 탁ML 당 1 × 107의 농도로 RP10 매체. 각 코팅 팬 (단계 1.3에서 준비) 판 소용돌이에 5 ㎖의 세포 현탁액을 추가하고 실온에서 30 분 동안 배양한다. 격렬하게 소용돌이 친다; 원뿔 튜브에 상층 액을 전송합니다. 판에게 RP10 매체를 사용하여 두 번 씻어 컬렉션 튜브에 상층 액을 풀. </li><li> 400 XG, 5 분 동안 4 ° C에서 포집 관을 원심 분리기. 대기음 액체 및 5 ㎖의 RP10 배지에서 세포 펠렛을 재현 탁. 새로운 패닝 플레이트에 세포 현탁액을 추가 소용돌이 및 RT에서 30 분 동안 배양한다. 세포가 접시를 씻어 원뿔 튜브로를 풀 수집합니다. TEC는이 충실하면, 정렬 버퍼 5 ML의 FACS에서 셀을 400 XG, 5 분 동안 4 ° C에서 세포를 스핀 다운 및 일시 중지합니다. </li></ol> 셀 정렬하여 TEC는의 6 정화 <ol><li> 혈구를 사용하여 세포를 세어 FACS 정렬 버퍼에 4 × 10 7 세포의 농도에서 세포 현탁액을 준비합니다. 항체 데 추가세포 용액에 4.4 절에 cribed 30 분 동안 4 ° C에서 회전 믹서에 부드럽게 세포를 배양한다. 염색 후, 씻어 FACS 정렬 버퍼의 ML 당 10 × 106 세포의 농도로 세포를 일시. </li><li> 정렬 100 μM 노즐을 통해 TEC 부분 집합은 형광 활성 세포 정렬을 사용. 정렬 세포는 RPMI-1640 (v / v)로 FBS) 30 %에 수집됩니다. 셀 정렬은 400 XG, 5 분 동안 4 ° C에서 원심 분리기 세포를 수행하고 다운 스트림 분석을 위해 준비되면. </li></ol>

<ol>
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Immunol. 119, 85-100 (1989).</li><li>Volkmann, A., Zal, T., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-presenting+cells+in+the+thymus+that+can+negatively+select+MHC+class+II-restricted+T+cells+recognizing+a+circulating+self+antigen.">Antigen-presenting cells in the thymus that can negatively select MHC class II-restricted T cells recognizing a circulating self antigen.</a> J. Immunol. 158, 693-706 (1997).</li><li>Yang, S. J., Ahn, S., Park, C. S., Choi, S., Kim, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+subpopulations+of+thymic+epithelial+cells+by+flow+cytometry+using+a+new+specific+thymic+epithelial+marker,+Ly110.">Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110.</a> J. Immunol. Method. 297, 265-270 (2005).</li><li>Gray, D. H., Chidgey, A. P., Boyd, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thymic+stromal+cell+populations+using+flow+cytometry.">Analysis of thymic stromal cell populations using flow cytometry.</a> J. Immunol. Method. 260, 15-28 (2002).</li><li>Gray, D. H., 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=Unbiased+analysis,+enrichment+and+purification+of+thymic+stromal+cells.">Unbiased analysis, enrichment and purification of thymic stromal cells.</a> J. Immunol. Method. 329, 56-66 (2008).</li><li>Williams, K. 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=Single+cell+analysis+of+complex+thymus+stromal+cell+populations:+rapid+thymic+epithelia+preparation+characterizes+radiation+injury.">Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury.</a> Clin. Transl. Sci. 2, 279-285 (2009).</li><li>Kanof, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+T+cell+subpopulations.">Purification of T cell subpopulations.</a> Curr. Protoc. Immunol. 7 (Unit 7.3), (2001).</li><li>Mage, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+T+cells+and+B+cells+using+panning+techniques.">Fractionation of T cells and B cells using panning techniques.</a> Curr. Protoc. Immunol. 3 (Unit 3.5), (1991).</li><li>Wekerle, H., Ketelsen, U. P., Ernst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+nurse+cells.+Lymphoepithelial+cell+complexes+in+murine+thymuses:+morphological+and+serological+characterization.">Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization.</a> The Journal of experimental medicin. 151, 925-944 (1980).</li><li>Kyewski, B. A., Kaplan, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoepithelial+interactions+in+the+mouse+thymus:+phenotypic+and+kinetic+studies+on+thymic+nurse+cells.">Lymphoepithelial interactions in the mouse thymus: phenotypic and kinetic studies on thymic nurse cells.</a> J Immuno. 128, 2287-2294 (1982).</li><li>Nakagawa, Y., 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=Thymic+nurse+cells+provide+microenvironment+for+secondary+T+cell+receptor+alpha+rearrangement+in+cortical+thymocytes.">Thymic nurse cells provide microenvironment for secondary T cell receptor alpha rearrangement in cortical thymocytes.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 20572-20577 (2012).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J. Immunol. Method. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of immunological method. 385, 23-34 (2012).</li></ol>

저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

프로토콜에서 중요한 단계는 흉선 간질 세포 (섹션 3)의 준비와 TEC는의 농축 (4 절)입니다. 이 적극 신선한 효소 용액마다 준비하고, 조직이 가능한 한 빨리 처리하는 것이 좋다. 풀링 thymi 들어, 효소 용액의 부피를 최적화 thymi의 수에 따라 요구된다. 모든 조직 잔사 처리 후에 남아 있으면, 더 효소액 및 배양 시간의 연장을 첨가하여 세포 수율을 증가시킬 수있다. TEC는의 농축, 상층 액의 수집을 완...

초기 T 세포 개발, 골수 조혈 줄기 세포 유래 다 분화능 전구 세포가 흉선의 피질 모집, T 계보에 대한 약속을 받아야하고 T 세포 전구체 1이된다. 매우 가변적 T 세포 수용체 1 전구 세포의 큰 풀을 형성 피질에서, T 세포 전구체 CD4 및 CD8 더블 네거티브 (DN) 흉선 세포 확장 및 미성숙 CD4 및 CD8 이중 양성 (DP) 흉선 세포로 분화. 만 DP 세포의 선택 MHC 제한 집합은 CD4 또는 CD8 하나의 긍정적 인 (SP) 흉선 세포가 흉선의 수질로 마이그레이션하고, 기능적 능력 성숙 T 세포에 긍정적 인 선택 2 불린다 이벤트를 차별화 6. 반면, 자동 반응 흉선 세포의 클론 부정적인 선택을 받아야하고 자기 공차 규제 T 세포로 변환, 또는 아직 명확하지 않은 목적을 위해 상피내 림프구로 전환, 세포 사멸을 통해 제거 3,7-10. 흉선에서, 흉선 간질 세포는 이러한 다양한 T 세포 발달의 운명 5,11,12에 대한 신호를 제공하는 고유의 미세 환경을 형성한다. 대뇌 피질의 TEC는 (cTECs) 및 수질의 TEC는 (mTECs), 수지상 세포, 대 식세포, 섬유 아세포, 혈관 내피 세포, 신경 크레스트에서 파생 된 혈관 주위 세포와 다른 중간 엽 세포 13 ~ 15 포함 - 흉선 간질 세포는 흉선 상피 세포 (TEC는)로 구성된다. 이들 중에서는 TEC는 T 세포의 발달 1,2,16,17 다양한 단계에 중요하다. 그러나, TEC는 격리 할 수있는 강력한 방법의 부족은 그 기능 (16)의 포괄적 인 이해를 방해하고있다. 특히 피질 전구 세포를 둘러싸 삼차원 네트워크를 형성 cTECs는 아직 확실하지 않은 이유로 양성 선택 13,18,19 필수적이다. 이전 연구는 대부분의 형태 학적 및 조직 학적 도구 (13)에 의존, TEC는의 이질성과 역할에 대한 단서를 제공 </sup&gt;. 최근 TEC 부분 집합의 고유 한 역할은 마우스 모델 12, 20에 유전 적 방법에 의해 해결되었다. TEC는을 분리하는 강력하고 재현 가능한 방법은 cTECs가 긍정적 인 선택을 어떻게 지원하는지 TEC 부분 집합, TEC 기능의 양적 및 질적 평가 및 메커니즘의 해명의 편견 특성을 달성하기 위해 필수적입니다. 때문에 그들은 그대로 장기에 형성 흉선에서 TEC는의 희소성과 긴밀한 상호 작용, TEC는의 분리 도전하고있다. 여기에 설명 된 프로토콜은 이전의 발견, 현재 이용 가능한 시약, 기법, 및 흉선 구조 및 기질 조성물의 지식에 기초한다. 거의 20 년 전, 몇 가지 절차는 트립신, 콜라게나 제와 디스 파제 등 다양한 효소가 소화하는 동안 사용 된에서 흉선 조직 21-27을, 해리하는 것으로보고되었다. 회색 등. 그들의 precedure 28에서와 효소를 비교하고 개선 메트 신고되었고 효소 칵테일 (29)의 여러 단계 소화 외경 (20), (30)을 널리 사용 하였다. 그러나,이 방법은 긴 준비 시간, 심지어 동일한 코호트 29,30 쥐에서 세포 수의 비율과 TEC는 최종 변수 복잡한 소화 단계 및 결과를 포함한다. 몇 년 전에, Liberase 연구 등급 효소, 고도로 정제 된 콜라게나 중성 프로테아제는 흉선 조직의 해리 (30)에 사용되기 시작 함유. 여기서 우리는 마우스 흉선 조직에서 실행 가능한 TEC는 높은 수를 산출 최적화 된 기계적 분리 절차 Liberase 소화 기반 프로토콜을, 설명합니다. . 해리 기질 세포를 풍부하게하기 위해, 이전의 연구는 밀도 구배 또는 자기 비드 분리 21,29를 사용하고 있습니다. 그러나 두 방법은 흉선 간질 세포, cTECs 29 특히 인구의 특정 집단의 손실 심각한 원인이된다. 세포 독성 제거 및 패닝 기법 <s&gt; (31, 32)까지 널리 면역 필드 12,31에 고갈 또는 림프구의 분리를 위해 사용되어왔다. 이러한 기술을 비교 한 결과, TEC는 우리의 농축을위한 전류 패닝 프로토콜을 세웠다. 농축 절차를 수행하는 동안 부드러운 상태가 적은 세포 죽음과 편견 증가 TEC 복구로 연결됩니다. 제 3 항에 기재된 절연 흉선 세포 현탁액을 직접 TEC 서브 세트와 수지상 세포의 확인 및 특성화를위한 유세포 분석 흐름에 적용될 수있다. 4 장에서는 사이토 다중 매개 흐름을 사용하여 TEC 하위 집합을 식별 할 수있는 간단하고 유용한 방법을 설명합니다. 정제 cTECs 또는 mTECs, TEC 농축 및 절차를 정렬 셀을 얻기 위해 추구 실험을 위해 제 5 및 6에서 찾을 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

흉선은 T 림프구 개발을위한 중요한 기관이다. T 세포 고집, 긍정적 및 부정적 선택을 선택 : 흉선 간질 세포, 흉선 상피 세포 (TEC는)은 T 세포 발달의 여러 단계에서 특히 중요하다. 그러나, 흉선에서 TEC는의 기능이 불완전하게 이해 상태를 유지합니다. 기사에서는 기계적 중단과 소화 효소의 조합을 사용하여 신선한 마우스 흉선에서 TEC 서브 세트를 분리하는 방법을 제공한다. 방법은 흉선 기질 세포 및 흉선 세포를 효율적으로 세포 - 세포 및 세포 - 세포 외 기질로부터 연결 해제 될 단일 세포 현탁액을 형성 할 수있다. 세포 격리하여, 다중 매개 유동 세포 계측법은 TEC는 수지상 세포의 확인 및 특성화에 적용될 수있다. TEC는가 흉선에서 희귀 세포 집단 때문에, 우리는 또한 풍부하고 흉선 세포, 흉선에서 가장 풍부한 세포 유형을 고갈하여 TEC는 정화 할 수있는 효과적인 방법을 설명한다. FOLLO세포 생존의 상실 TEC는 정제 과정을 최소화 할 수 있도록 날개가 농축, 세포 정렬 시간은 감소 될 수있다. 정제 된 세포는 리얼 타임-PCR, 웨스턴 블롯 및 유전자 발현 프로파일 링 같은 다양한 다운 스트림 분석에 적합하다. 프로토콜은 TEC의 기능 연구뿐만 아니라 시험 관내 T 세포 재구성의 발전을 촉진 할 것이다.

수득 된 세포 현탁액은 흉선 세포, 조혈 유래 기질 세포 및 비 - 조혈 기질 세포로 구성 3 절에서 설명한 바와 같이이 프로토콜을 사용하여, 흉선 오르간 성인 마우스 (섹션 2) 및 흉선 세포 현탁액으로부터 제거 제조 하였다. CD45는 조혈 팬 마커가 흉선 세포 및 대 식세포와 수지상 세포로 조혈 기질 세포 모두에서 발현이다. 반면, TEC는 조혈 기원하지 않으며 CD45 15을 표명하지 아니합니다. ?...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

여기에서 우리는 분리, 식별 및 마우스 흉선 상피 세포 (TEC는) 정화용 효율적인 방법을 설명합니다. 프로토콜은 정상 T 세포의 발달, 흉선 기능 장애, 및 T 세포 재구성을위한 흉선 기능의 연구에 활용 될 수있다.

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-identification-purification-murine-thymic-epithelial

Research

JoVE Journal

Immunology and Infection

31.7K 조회수.  University of Minnesota.  여기에서 우리는 분리, 식별 및 마우스 흉선 상피 세포 (TEC는) 정화용 효율적인 방법을 설명합니다. 프로토콜은 정상 T 세포의 발달, 흉선 기능 장애, 및 T 세포 재구성을위한 흉선 기능의 연구에 활용 될 수있다. 

비디오: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

마우스의 응용은 M 세포에 의해 박테리아 이해를 평가하는 Peyer의 패치 장 루프 분석 출혈도 잡았

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

연구

면역학 및 감염병학

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

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 of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

망막 색소 상피 세포에 탐식 분석 실험에 대한 돼지 또는 소 광 수용체 외부 세그먼트의 대규모 정화

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

분리 및 다른 세 쥐 흉선 이식의 이식.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

EAK 16-II와 FACS 정화 흉선 상피 세포의 3 차원 집계를 홍보 / EAKIIH6 자기 조립 하이드로 겔을

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.