우리는 셀 정렬을 위해 아론 레이를 (에모리 대학 소아과학과와 애틀랜타 흐름 코어의 어린이 건강 관리) 감사합니다. 이 작품은 TLD에 NIH 교부금 AA01787001, Crohn의 및 미국의 대장염 재단의 경력 개발 수상 및 에모리 - Egleston 아동 연구 센터 종자 교부금에 의해 지원되었다

1. 해부와 창자 상피 세포의 분리 시약 및 장비의 준비 : <ul><li> 실온에 따뜻한 칼슘 2 + / MG 2 + 프리 PBS (CMF PBS). </li><li> 5% FBS (CMF HBSS / FBS)와 실온에 2mM EDTA (에틸렌 다이아 민 테트라 초산)과 따뜻한 칼슘 2 + / MG 2 + 무료 HBSS. </li><li> 37까지 따뜻한 궤도 흔드는 ° C. </li></ul> 참고 : 1.7-1.1 단계는 세포 사망의 범위를 최소화하고 최대 세포 수율을 달성하기 위해 최대한 빨리 수행해야합니다. <ol><li> 복부 및 흉부 향해 CO 2 챔버 및 스프레이 70 % 에탄올에 쥐를 안락사시켜야. </li><li> 가위와 다시 껍질 복막을 노출하기 위해 피부와 복부의 중간에 작은 수평 절개를합니다. </li><li> pyloric 괄약근에서 절단하여 상부 소장에서 배를 분리 진행합니다. 집게와 장간막을 떨어져 애타게과에 다시 절단ileo-cecal 밸브가 대장에서 전체 소장을 풀고있다. 항문 직전에 상처를 만들고 대장은 무료이며 때까지 다시 장간막을 떨어져 애타게. </li><li> 길이 방향 가위를 사용하여 콜론을 열고 잘라 실온에서 CMF PBS의 창자 루멘에서 배설물 내용과 점액을 씻어. </li><li> macroscopically 작은 창자의 안티 mesenteric 표면을 따라 Peyer의 패치를 해부하고 길이 방향 작은 창자를 오픈 잘라 가위와 집게를 사용하십시오. </li><li> 실온에서 CMF PBS의 지저분한 내용과 점액의 작은 창자의 루멘 씻으십시오. </li><li> 별도로 사전 예열 CMF HBSS / FBS와 2mM EDTA (에틸렌 다이아 민 테트라 초산)의 30 ML을 포함하는 별도의 50 ML 원뿔 튜브에 약 1.5 cm의 조각과 장소에 작은 / 큰 창자를 잘라. 50 ML 원뿔 튜브 당 1 이상 소장을 추가하지 마십시오. </li><li> 수평 ° C. &lt;37시 20 분 동안 궤도 흔드는로 각각 50 ML 원뿔 튜브를 삽입 및 250 rpm으로 위해 악수/ 리&gt; <li> 폐기물 양동이를 통해 하나의 메쉬 와이어 스트레이너를 삽입하고 소장의 1.5 cm 조각을 복구를 통해 각각 50 ML 원뿔 튜브의 내용을 부어 2로 미리 예열 CHBSS / FBS의 30 ML을 포함하는 별도의 50 ML 원뿔 튜브에 넣어 MM EDTA (에틸렌 다이아 민 테트라 초산). </li><li> 1.8 반복합니다. </li></ol> 2. 조직 소화와 창자 세포의 분리 시약 및 장비의 준비 : <ul><li> Collagenase 솔루션 : 1.5 MG / ML 타입 VIII Collagenase는 DNase 나의 40 μg / ML로 미리 예열 CMF HBSS / FBS에 녹아있는 </li><li> 사전 예열 CMF HBSS / FBS와 2mM EDTA (에틸렌 다이아 민 테트라 초산). </li><li> 37 미리 예열 궤도 흔드는 ° C. </li><li> 얼음처럼 차가운 CMF HBSS / FBS. </li></ul><ol><li> 흔들림의 두 번째 라운드 후, 스트레이너를 통해 각각 50 ML 원뿔 튜브의 내용을 부어하고 작은 플라스틱으로 소장의 1.5 cm 조각을 전송 종이 타월을 사용하여 여분의 미디어를 멀리 dabbing 후 보트를 달다. </li><li> 신속하게 1을 말하다소장의 0.5 cm 조각 체중 보트에 직접 가위를 사용하고 collagenase 용액의 20 ML에 다진 소장을 추가합니다. 가로 37에 10-20 분 동안 200 rpm으로 궤도 흔드는 및 다이제스트로 각각 50 ML 원뿔 튜브를 삽입 ° C.는 최적화에 관한 아래의 논의를 참조하십시오. </li><li> 직접 50 ML 원뿔 튜브에 100 μm의 셀 스트레이너를 통해 언제 나머지 장 조직 및 필터의 철저한 분리를 보장 간략히 소용돌이. </li><li> CMF HBSS / FBS 및 4에서 5 분 1,500 rpm으로 원심 ° C. 각각 50 ML 원뿔 튜브 톱 오프 고체 펠렛은 원심 분리 후 콜론 샘플에 대해 관찰되지 않을 경우 샘플은 3.5 분 동안 다시 centrifuged해야합니다. 한번 더이 세차 단계를 반복합니다. </li><li> 뜨는을 붓고 얼음처럼 차가운 CMF HBSS / FBS 및 장소 샘플 얼음에 세포 펠렛을 resuspend. </li><li> 고속 셀 sorti위한 자성-비드 농축을위한 FACS 수집 / 분석 또는 제 4 항 3으로 진행합니다NG. </li></ol> 3. DCS 및 Macrophages의 멀티 컬러 흐름 Cytometric 분석을위한 항체 염색법 시약 및 장비의 준비 : <ul><li> 얼음처럼 차가운 CMF PBS. </li><li> 얼음처럼 차가운 염색법 버퍼 (CMF PBS가 + 5퍼센트 FBS). </li><li> 죽었 / LIVE 고칠수 아쿠아 죽은 세포 얼룩 키트를 사용하여 1:1000 희석에 얼음처럼 차가운 CMF PBS에 얼룩 죽은 세포를 준비합니다. </li><li> CD45-PerCP, CD103-PE, CD11c-APC, MHC-II (IA B) - 알렉사 형석 700 CD11b 다음 형광 라벨이 표시된 단클론 항체 (mAbs) 심심풀이 염색법 버퍼에 추가하여 항체 염색법 칵테일을 준비 - eFluor 450 F4/80-PE-Cy7. </li></ul><ol><li> 5 ML의 폴리스티렌 왕복 하단 (FACS) 튜브에 세포를 전송합니다. </li><li> 얼음처럼 차가운 CMF PBS로 두 번 세포를 씻으십시오. </li><li> 죽은 세포로 품어 샘플은 어두운 얼음 속에서 15 분 동안 얼룩. </li><li> 얼음처럼 차가운 CMF PBS로 두 번 세포를 씻으십시오. </li><li> 얼음처럼 차가운 염색법의 2.4G2 anti-FcγRIII/II있는 블록 세포얼음에 10 분 대한 버퍼. </li><li> 얼음처럼 차가운 염색법 버퍼에 세포를 씻으십시오. </li><li> 어두운 얼음 속에서 20 분 대한 항체 염색법 칵테일과 샘플을 품어. </li><li> 두번 심심풀이 염색법 버퍼로 세포를 씻어과 얼음처럼 차가운 염색법 완충액 400 μl에 샘플을 resuspend하고 FACS 튜브 40 μm의 필터 캡을 통과. </li><li> 제 5 및 그림 1에 게이팅 전략에 의해 정의된대로 LSR II cytometer (BD)에 샘플을 습득. </li></ol> 4. 소장의 DCS 및 Macrophages의 농축 시약 및 장비의 준비 : <ul><li> 얼음처럼 차가운 염색법 버퍼 (CMF PBS가 + 5퍼센트 FBS). </li></ul><ol><li> 제조 업체의 지침에 따라 CD11b와 CD11c 맥에서 구슬있는 단계를 2.5에서 얻은 단일 세포 현탁액을 품어. </li><li> 원심 분리에 의해 다음에 얼음처럼 차가운 염색법 버퍼로 세포를 씻으십시오. </li><li> 뜨는을 무시하고 1 ML 심심풀이 염색법 버퍼에 세포 펠렛을 resuspend그리고 40 μm의 셀 스트레이너 다음에 100 μm의 셀 스트레이너 통과. </li><li> 맥에서 LS 자기 컬럼을 이용하여 긍정적인 선택에 의해 자성 비드 연결된 세포에 대한 더욱 돋보이게합니다. </li><li> 단계 4.2 반복하고 뜨는 버리고. </li><li> 와 같은 단계를 3.7에서 설명한 표면 마커 mAbs과 세포를 품어. </li><li> 얼음처럼 차가운 염색법 버퍼로 두 번 마그네틱 비드 - 농축 세포를 씻으십시오. 나트륨 azide없이 500 μL 심심풀이 염색법 버퍼에 셀 알약을 Resuspend하고, FACS 튜브로 40 μm의 셀 스트레이너 통과. </li><li> 창자 DC 및 / 또는 관심있는 대식 세포의 하위 집합을 정렬하는 BD ARIA II 세포 분류기에서 FACS-분류로 진행합니다. </li></ol> 5. LP APCs위한 게이팅 전략 참고 : 흠없는 창자 세포가 긍정과 부정 인구를 구분하기 위해 성문의 적절한 배치를 지원하기 위해 부정적인 제어로 활용됩니다 유의하시기 바랍니다. <ol><li> 그림에 나타난 바와 같이1 도트 계획을 작성하고 이중어 행사의 배제 (그림의 1B 및 C) 다음에 얼룩 죽은 세포 (그림 1A) 양성 반응 세포를 제외할 수 있습니다. 그런 다음, 전달 및 사이드 분산형는 (그림의 1D) 파편을 제외해야하기에 따라 관심의 세포에 게이트. </li><li> CD45의 다른 점 줄거리와 더 게이트 만들기 +와 아이오와 B + phenotypically APCs (그림 1E)를 특징 세포. </li><li> 별도의 도트 줄거리에서 CD11b 및 특정 DC 및 대식 세포의 하위 집합을 (.; 그림 1 층 R1, R2 및 R3) 구별하는 CD11c 표현에 대해 분석합니다. R1의 세포는 CD11c + CD11b입니다 / 무뎌 - 세포. 지역, R2는 이러한 세포가 R1의 세포와 같이 CD11c 위해 표면 표현의 유사한 수준을 가지고 있지만 R2의 세포도 급행 CD11b 그러한 delineated있다. - 이후 R3 지역은 CD11b +를 그리고 / 무딘 CD11c있는 세포를 위해 지정되어 있습니다. </li><li>지역 R1, R2, 그리고 대식 세포 및 DC 인구를 차별화하기 위해 R3 F4/80에 대한 그리고 CD103 표현, 각각을 alyze. CD11c + CD11b / 무딘 - αE 인테, CD103, 그리고 F4/80의 낮은 수준 (그림 1G)의 R1 명시적인 높은 수준의 세포. CD11b + CD11c + R2의 세포는 CD103과 F4/80 (그림 1H)들의 dichotomous 표현을 바탕으로 DCS 및 macrophages 모두 구성되어 있습니다. 마지막으로, CD11b + CD11c 무딘 / - R3의 세포가 F4/80 +와 CD103의 phenotypic 프로파일을 기반으로 macrophages를 구성 - (그림 1I) 16. </li></ol> 6. 대표 결과 <img alt="그림 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> 1 그림. 창자 DCS 및 macrophages를위한 게이팅 전략. 죽은 세포 ()와 doublets (B와 C)은 첫번째 작은 intesti 다음 분석에서 제외하고 있었다nal 세포 전달 따라 게이트와 사이드 분산형 (D) 였는데, APCs는 CD45과 같이 정의되었다 + 아이오와 B + (E). Macrophages와 DC가이 CD11b와 CD11c (F)의 표현에 의해 확인되었다. CD103과 사전 문이 R1 (G), R2 (H)와 인구 (I) R3에서 전지 F4/80 식을 분석되었다. <img alt="그림 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> 그림 2. 세포 수율 및 항체 염색법 품질은 소화 시간에 따라 달라집니다. CD11b와 CD11c 염색법 패턴 이하 - (A, D)의 총 세포 수율, 최적 - (B, E) 또는 오버 소화는 (C, F) 장 조직. 창자 세포는 C57BL / 6 마​​우스 소장으로부터 격리되었고 DCS 및 macrophages는 BD LSR II에 FACS로 분석되었다. 전압 및 보상은 흠없는 및 단일 fluorochrome 묻은 splenocytes를 사용하여 설정되었다. 죽은 세포 (그림 1A) 및 doublets은 (그림의 1B 및 C) 처음부터 제외되었습니다분석. 관심있는 세포 그 다음 전달하고 따라 분석되었다 CD45에 게이팅 뒤에 사이드 분산형 (그림의 1D) + 및 IA B + 세포 (그림 1E). 이후 CD11b와 CD11c 표현은 CD45 사이에 평가되었다 + 아이오와 B 세 지역 (R1, R2 및 R3., 그림 1 층) 윤곽을 그리다하기 + 세포. 세 지역에서 CD103과 F4/80 표현은 각각 DCS 및 macrophages 구별 평가되었다. CD11c + CD11b 무딘 / - R1의 세포가 αE 인테, CD103, 그리고 F4/80의 낮은 수준 (그림 1G)의 높은 수준을 보였다. CD11b + CD11c + CD11b + CD11c / 무딘 동안 R2의 세포는 CD103과 F4/80 (그림 1H)들의 dichotomous 표현을 바탕으로 DCS 및 macrophages 모두 구성되어 있었다 - R3의 세포 F4 /의 phenotypic 프로파일을 기반으로 macrophages를 구성 80 +와 CD103 - ( <str긴 사연&gt; 그림. 1I) 16. R3 게이트에서 R2 게이트와 macrophages 내에서 Macrophages 유사한 순방향 및 사이드 산란 특성을 가지고 있으며 CD11c 표현하여 구별할 수 있습니다. 이 하위 집합의 기능적인 이분법은 불완전하게 이해 남아 있습니다. 총 세포 수율 및 CD11b와 CD11c의 표현에 대한 조직 소화 기간의 관계는 그림 2에 설명된됩니다. 3 분 (이하 - 소화)를 소화했다 장 조직은 낮은 총 휴대폰 번호 (그림 2D) 그래서 몇 DCS 및 특성에 사용할 수 macrophages (그림 2A)를 굴복. 11 분 용 티슈는 소화 CD11b와 CD11c 높은 수준을 표명하고 (그림 2C) phenotypically 독특했다 DCS 및 macrophages의 인구 라이브 셀 (그림의 2E)의 강력한 수확량을 생산했다. 대조적으로 50 분 (오버 소화)에 대한 소화가 유사한 세포 수율으로 이어지는 경우 공동최적 소화 (그림 2E와 F)에 mpared 그러나, CD11b와 CD11c를 사용하여 서로 다른 세포 집단의 묘사는 (그림 2C) CD11c 줄었의 표현으로 더 애매한되어 죽은 세포의 숫자 (데이터가 표시되지 않음) 증가했다.

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L., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+intestinal+immune+regulation.">Dendritic cells in intestinal immune regulation.</a> Nat. Rev. Immunol. 8, 435-446 (2008).</li><li>Kelsall, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding+the+phenotype+and+function+of+intestinal+dendritic+cells+and+macrophages.">Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages.</a> Mucosal Immunol. 1, 460-469 (2008).</li><li>Pulendran, B., Tang, H., Denning, T. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1-mediated+dendritic+cell+access+to+the+intestinal+lumen+and+bacterial+clearance.">CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance.</a> Science. 307, 254-258 (2005).</li><li>Milling, S. W., Cousins, L., MacPherson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+DCs+interact+with+intestinal+antigens.">How do DCs interact with intestinal antigens.</a> Trends Immunol. 26, 349-352 (2005).</li><li>Bilsborough, J., Viney, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell:+its+role+in+intestinal+inflammation+and+relationship+with+gut+bacteria.">The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria.</a> Gut. 52, 1522-1529 (2003).</li><li>Medina-Contreras, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CX3CR1+regulates+intestinal+macrophage+homeostasis,+bacterial+translocation,+and+colitogenic+Th17+responses+in+mice.">CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice.</a> J. Clin. Invest. 121, 4787-4795 (2011).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Specializations+of+Intestinal+Dendritic+Cell+and+Macrophage+Subsets+That+Control+Th17+and+Regulatory+T+Cell+Responses+Are+Dependent+on+the+T+Cell/APC+Ratio,+Source+of+Mouse+Strain,+and+Regional+Localization.">Functional Specializations of Intestinal Dendritic Cell and Macrophage Subsets That Control Th17 and Regulatory T Cell Responses Are Dependent on the T Cell/APC Ratio, Source of Mouse Strain, and Regional Localization.</a> J. Immunol. , 187-733 (2011).</li><li>Kim, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nod2+sensor+promotes+intestinal+pathogen+eradication+via+the+chemokine+CCL2-dependent+recruitment+of+inflammatory+monocytes.">The Nod2 sensor promotes intestinal pathogen eradication via the chemokine CCL2-dependent recruitment of inflammatory monocytes.</a> Immunity. 34, 769-780 (2011).</li><li>Schulz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+CD103+,+but+not+CX3CR1+,+antigen+sampling+cells+migrate+in+lymph+and+serve+classical+dendritic+cell+functions.">Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions.</a> J. Exp. Med. 206, 3101-3114 (2009).</li><li>Jaensson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestinal+CD103++dendritic+cells+display+unique+functional+properties+that+are+conserved+between+mice+and+humans.">Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans.</a> J. Exp. Med. 205, 2139-2149 (2008).</li><li>Uematsu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+humoral+and+cellular+gut+immunity+by+lamina+propria+dendritic+cells+expressing+Toll-like+receptor+5.">Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5.</a> Nat. Immunol. 9, 769-776 (2008).</li><li>Schenk, M., Bouchon, A., Seibold, F., Mueller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1--expressing+intestinal+macrophages+crucially+amplify+chronic+inflammation+in+experimental+colitis+and+inflammatory+bowel+diseases.">TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases.</a> J. Clin. Invest. 117, 3097-3106 (2007).</li><li>Sun, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestine+lamina+propria+dendritic+cells+promote+de+novo+generation+of+Foxp3+T+reg+cells+via+retinoic+acid.">Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid.</a> J. Exp. Med. 204, 1775-1785 (2007).</li><li>Kamada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormally+differentiated+subsets+of+intestinal+macrophage+play+a+key+role+in+Th1-dominant+chronic+colitis+through+excess+production+of+IL-12+and+IL-23+in+response+to+bacteria.">Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria.</a> J. Immunol. 175, 6900-6908 (2005).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++Th+cells+resembling+regulatory+T+cells+that+inhibit+chronic+colitis+differentiate+in+the+absence+of+interactions+between+CD4+and+class+II+MHC.">CD4+ Th cells resembling regulatory T cells that inhibit chronic colitis differentiate in the absence of interactions between CD4 and class II MHC.</a> J. Immunol. 171, 2279-2286 (2003).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="그림 3"/> 그림 3. 세포 수율 및 표면 항원 표현의 최적화에 중요한 요인. 세포 수율 및 표면 항원 표현이 직접적으로 조직 소화 기간의 영향을받는, collagenase의 구체적인 특성, 닦지 조직의 정도와 염증의 유무, 이는 조직 무결성 및 cellularity 영향을 미칠 수 있습니다. 불충 분한 조직 소화가 분석을위한 세포의 소수가 발생할 수도 있지만 장기 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 1X PBS, CA 2 + -와 MG 2 + 프리 </td><td></td><td></td><td></td></tr><tr><td> 페놀 레드와 행크의 균형 소금 용액 (HBSS) </td><td> 피셔 과학 </td><td> SH3001603 </td><td></td></tr><tr><td> 나트륨 중탄산염 </td><td> 시그마 </td><td> S6014 </td><td></td></tr><tr><td> 0.85 % NaCl에서 1M HEPES </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> 애틀랜타 생체 </td><td> S11150H </td><td> 히트-inactivated </td></tr><tr><td> 0.5M EDTA (에틸렌 다이아 민 테트라 초산) (산도 8.0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> CollagenasE 타입 VIII </td><td> 시그마 </td><td> C2139 </td><td></td></tr><tr><td> DNase I </td><td> 로슈 </td><td> 14785000 </td><td> 증권 솔루션 : 100mg/ml </td></tr><tr><td> 405 나노미터 여기에 죽은 / LIVE 고칠수 아쿠아 죽은 세포 얼룩 키트 </td><td> Invitrogen </td><td> L34957 </td><td> 1:1000에서 사용 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557,235 </td><td> 1:100에서 사용 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557,495 </td><td> 1:100에서 사용 </td></tr><tr><td> FcγRIII / II mAb (2.4G2) </td><td> BD </td><td> 553,141 </td><td> 1:200에​​서 사용 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> 1:100에서 사용 </td></tr><tr><td> MHC-II (I-AB) - 알렉사 형석 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> 1:100에서 사용 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> 1:200에​​서 사용 </td></tr><tr><td> F4/80-PE-Cy7 mAb (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b microbeads </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microbeads </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ML 원뿔 튜브 </td><td> BD 팔콘 </td><td> 352,098 </td><td></td></tr><tr><td> 단일 메쉬 와이어 스트레이너 </td><td> Chefmate </td><td></td><td></td></tr><tr><td> 스몰 보트를 달다 </td><td> 피셔 과학 </td><td> 08-732-116 </td><td></td></tr><tr><td> 100 μm의 셀 스트레이너 </td><td> BD 팔콘 </td><td> 352,360 </td><td></td></tr><tr><td> 40 μm의 셀 스트레이너 </td><td> BD 팔콘 </td><td> 35,2340 </td><td></td></tr><tr><td> 5 ML의 폴리스티렌 왕복 하단 튜브 </td><td> BD 팔콘 </td><td> 352,235 </td><td> 1:100에서 사용 </td></tr><tr><td> MaxQ 4450 benchtop 궤도 흔드는 </td><td> 써모 과학 </td><td></td><td></td></tr><tr><td> LS 맥에서 칼럼 </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

소장 내에서 concomitantly 남아 침입 병원균의 1,2으로 염증 반응을 마운트 태세를하면서 공생 식물 및 식품 항원으로 허용 오차를 추진에 관여하고 타고난 및 적응 면역 세포의 독특한 인구 거주. 항원 제시 세포, 특히 DCS 및 macrophages은 감각 능력을 통해 장내 면역 항상성을 유지하는 중요한 역할을 적절하게 microbiota 3-14에 대응. 창자 DCS 및 macrophages의 효율적인 분리는 이러한 세포의 표현형 및 기능을 특성화의 중요한 단계입니다. DCS 및 macrophages를 포함한 장내 면역 세포를, 분리 많은 효과적인 방법은 6,10,15-24 설명되어 있지만, 많은 사람들이 부정적인 세포 표면 항원 발현, 세포 생존 및 / 또는 세포 수율에 영향을 미칠 수있다 긴 digestions 시간에 의존. viabl 다수의 신속한 격리를위한 여기서는 세부 방법론을E, 창자 DCS 및 macrophages. 창자 DCS 및 macrophages의 Phenotypic 특성화 직접 멀티 컬러 흐름 cytometric 분석을위한 구체적인 형광 라벨이 표시된 단클론 항체와 격리된 창자 세포를 더럽히는 것에 의해 수행됩니다. 또한, 매우 순수 DC 및 대식 세포의 인구는 셀 정렬 뒤에 CD11c 및 CD11b 자기가 활성화된 세포 분류 구슬을 이용한 기능성 연구에 대해 격리됩니다.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

여기서는 자세하게 마우스 장 돌기 세포 (DC가)와 macrophages의 급속한 분리를위한 방법론. 창자 DCS 및 macrophages의 Phenotypic 특성은 세포 정렬 뒤에 자석 구슬 농축은 기능적 연구를위한 매우 순수 인구를 얻을하는 데 사용하는 동안 멀티 컬러 플로우에게 cytometric 분석을 사용하여 수행됩니다.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

isolation-characterization-dendritic-cells-macrophages-from-mouse

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Immunology and Infection

38.9K 조회수.  Emory University. 여기서는 자세하게 마우스 장 돌기 세포 (DC가)와 macrophages의 급속한 분리를위한 방법론. 창자 DCS 및 macrophages의 Phenotypic 특성은 세포 정렬 뒤에 자석 구슬 농축은 기능적 연구를위한 매우 순수 인구를 얻을하는 데 사용하는 동안 멀티 컬러 플로우에게 cytometric 분석을 사용하여 수행됩니다.

비디오: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

마우스 폐에 돌기 세포의 분리

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

연구

면역학 및 감염병학

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T 세포는 수지상 세포에서 Transinfection에 의해 박테리아를 캡처

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

이미징 유동 세포 계측법에 의해 인간 단핵구 유래 수지상 세포의 특성 : 두 단핵구 격리 프로토콜 사이의 비교

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

유동 세포 계측법에 의해 쥐의 신장에서 대 식세포의 표현형 특성

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

마우스 골수에서 형질 수지상 세포의 정화를위한 형광 활성화 셀 정렬

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.