우리는 바이오 안전성 수준이 샘플에서 기본 손쥐 AECII를 정렬에 기술 지원을위한 M. Höxter 감사드립니다. 이 작업은 DB에 독일 연구 재단 (DFG) (SFB587, TP B12 및 BR2221/1-1)와 하노버 바이오 메디컬 연구 학교 (DFG GSC 108)에서 AA에이고의 보조금에 의해 지원되었다. DB는 대통령의 이니셔티브 및 계약 번호 W2/W3-029에 따라 독일어 연구 센터의 Helmholtz 협회 (HGF)의 네트워킹 기금에서 지원됩니다.

필요한 시약 및 재료에 관한 자세한 내용은 아래의 프로토콜의 끝 부분에 다음 표에 나열되어 있습니다. 작업을 시작하기 전에, 4 dispase의 ML과 ° C 물 목욕에 37 미리 따뜻하게을 포함하는 15 ML 튜브를 (마우스 하나씩)을 준비합니다. 가열 블록에서 곧 95-1% 낮은 용해 아가로 오스 (물)의 작은 aliquots를 가열 ° C 액화 때까지 사용 할 때까지 45 ° C에이어서 좋아. 1. 마우스 폐의 작성 <ol><li> CO 2 질식하여 마우스를 희생. 참고 : 성공적으로 수행 될 수 없습니다와 같은 액체와 폐의 설치로, 프로토콜의 단계를 수행하므로이 기관을 손상하므로 자궁 경부 전위를 수행하지 마십시오. nociceptive 반사 손실을 보장합니다. </li><li> 에탄올로 마우스를 뿌려서 몸의 복부 중간 선을 따라 긴 몫을합니다. 복부 털과 피부를 당겨하고 이후도 조심스럽게 잘라 복막을 제거합니다. </li><li> 을 ...에게서 피를 뽑다왼쪽과 오른쪽 경정맥 정맥 (베나 jugularis)뿐만 아니라 신장 동맥 (Arteria renalis)를 절단하여 마우스. 조직과 흐르는 혈액을 제거합니다. </li><li> 조심스럽게 다음 찔린하고 늑골을 멀리 절단하여 마음과 폐를 노출 할 수있는 피임기구를 제거합니다. 폐를 손상하지 특별히 조심하고. </li><li> 추위 인산로 가득 26G 캐뉼라와 10 ML의 주사기로 생리 (PBS), 찔린 심장의 우심실은 버퍼와 혈액의 무료까지 PBS로 폐를 perfuse. </li><li> 기관을 폭로 할 수있는 침샘을 잘라 제거합니다. 또한주의 깊게 호흡 관을 둘러싼 근육을 잘라. </li><li> 기관에 22G 내주하시는의 캐뉼라를 삽입하고, 바늘을 제거하고 폐에 대한 플라스틱 카테터를 밀어. 기관 및 카테터 주변 원사의 작은 조각을 매는하여 카테터를 해결했습니다. </li></ol> 2. 폐 조직의 효소 소화 원하는 경우, bronchoalveolar 세척 유체 샘플 수다음 단계 전에 PBS 또는 매체 기관에 삽입 된 카테터를 통해 폐를 플러싱으로 준비했습니다. <ol><li> 2 ML의 주사기를 사용하여주의 깊게 모든 엽이 완전히 확장되도록 카테터를 통해 폐에 dispase 2 ML (4 ML의 나누어지는에서)의 최대 볼륨을 심어. 액화 아가로 오스의 0.5 ML를 포함하는 1 ML의 주사기와 주사기를 교환하고 또한 폐에 심어. </li><li> 아가로 오스의 다시 흐름을 방지하고 즉시 실험실 티슈과 얼음으로 폐를 커버 할 수있는 카테 테르에 주사기를 둡니다. 몇 분의 아가로 오스 젤을 보자. </li><li> , 휴지, 얼음, 주사기와 카테터를 제거 기관 및 소비세 폐, 심장과 가슴에서 thymus을 잘라. 그런 다음 PBS와 접시에 폐를 씻어 심장, thymus와 나머지 기관을 제거합니다. </li><li> dispase의 나머지 두 ML에 폐를 넣고 45 분 동안 실온에서 알을 품다. </li></ol> 3. 폐의 작성세포 현탁액 <ol><li> dispase에서 폐를 제거하고 DMEM 매체 100 μl DNase에 anti-CD16/32 항체의 7 ML (1 μg / ML 최종 농도)를 포함하는 요리로 전송. </li><li> 집게를 사용하여 완전히 떨어져 당겨 폐 조직을 분해하고 부드럽게 200 RPM으로 설정 로커에 흔들 동안 실온에서 10 분에 품다. 원하는 경우, 세포 현탁액은 다음 단계까지 4 ° C에 저장할 수 있습니다. </li><li> 100 μm와 이후 70 μm, 48 μm 및 30 μm 구멍이있는 첫번째 나일론 meshes를 통해 세포 현탁액을 필터링 할 수 있습니다. 세포의 손실을 최소화하고 수익을 극대화 할뿐만 아니라 DMEM으로 튜브 철저히 요리 각 메쉬를 씻어하는데주의를 기울여야. 당신이 샘플을 풀링하는 경우, 이것은 이후 같은 필터를 통해 한 그룹의 쥐에서 세포 현탁액을 통과하여 여기에 달성 될 수있다. 막힘의 경우 필터를 갱신. </li><li> 볼륨에 따라 하나 이상의 50 ML 튜브로 여과를 전송하고160 XG에서 15 분, 4 ° C.에 대해 원심 분리기 표면에 뜨는을 제거합니다. </li><li> 2 ML 적혈구 용해 완충액 (0.15 M NH 4 망할 CIA, 0.01 M KHCO 3, 0.1 MM EDTA (에틸렌 다이아 민 테트라 초산), 산도 7.2)에 세포 펠렛을 Resuspend 빠르게 DMEM 매체 13 ML의 추가에 의해 용해을 해지 할 수 있습니다. 160 XG에서 12 분, 4 ° C.에 15 ML 튜브와 원심 분리기에 솔루션을 전송 </li></ol> 4. 흐름 Cytometric 셀 정렬에 대한 항체 염색법 <ol><li> 유동 세포 계측법에 적합한 dilutions에 DMEM 매체에 준비 3 ML 기본 항체 칵테일에 세포를 Resuspend. (항체는 이전에 100 μl의 볼륨에 1 개 10 6 splenocytes 얼룩으로 최적의 작업 dilutions을 위해 titrated되었습니다. 사용 다섯 마우스까지에서 풀링 된 세포에 대한 사용 항체 각각에 대한 최적의 농도를 포함하는 차 항체 칵테일 3 ML. 더 많은 생쥐가 하나 샘플로 풀링하는 경우)의 볼륨을 조정합니다. </li><li> 착색를 들어, 어둠 속에서 10 분 동안 세포를 배양4 번 ° C. </li><li> 12 160 XG에서 분, 4 DMEM 매체와 원심 분리와 함께 15 ML 튜브를 작성하여 세포를 씻으 ° C. </li><li> 경우 uncoupled 또는 biotinylated 항체는 4.1에 사용되었습니다 표면에 뜨는을 제거하고 2 세포를 resuspend - 3 ML 보조 항체 칵테일. 어둠 속에서 4에 10 분 ° C에 청바지. </li><li> 160 XG에서 12 분, 4 ° C.에 DMEM과 원심 분리로 관을 작성을 통해 세포를 씻으십시오 </li><li> 셀 - 정렬의 튜브에 필터 50 μm을 통해 DMEM 및 사전 필터의 1 ML에있는 세포를 Resuspend. 선택 사항 : 폐 세포 현탁액의 전체 전화 번호를 얻기 위해 셀을 계산합니다. </li></ol> 5. 셀 - 정렬 <ol><li> 정렬하기 전에 vortexing하여 세포를 섞는다. AECII의 세포 분류에 100 μm의 노즐을 사용합니다. 칼집 유체 압력뿐만 아니라 레이저 방출 및 검출 파장은 흐름 cytometric 셀 정렬뿐만 아니라 antib에 사용 된 fluorochromes에 사용되는 악기에 따라 달라집니다ody 착색 절차. </li><li> DMEM을 포함하는 관으로 착색 및 정렬에 사용되는 fluorochromes에 대한 부정적인있는 SSC 높은 세포에서 게이트. (SSC-A) 창 (에 게이팅 전략의 세부 정보를 확인하는 doublets 또는 셀을 합산에서 제외하는 분산 형 높이 (FSC-H) 대 면적 (FSC-A)와 옆으로의 분산 높이 (SSC-H) 대 면적 앞으로 사용 1)을 그림. </li><li> 20 280 XG에서 분, 4 정렬 셀 원심 분리기를 수집하기 위해 ° C. </li><li> 후속 분석을 위해 필요한 문화 중간이나 버퍼에있는 셀을 Resuspend. </li></ol>

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관심 없음 충돌이 선언 없습니다.

유동 세포 계측법에 의한 손쥐 AECII의 절연에 대한 프로토콜은 기능과 분자 연구의 모든 범위의 마우스 폐에서 기본 셀을 액세스의 급속한 방법을 제공합니다. 설명 절차는 RNA 분리 (그림 2B 참조) transcriptome 연구로 직접 후속 분석을위한 번호 충분 AECII의 높은 가능한 순수 인구를 산출. 기능 어플리케이션의 경우, AECII 에어컨, 중간 또는 공동 문화 실험의 생성 예 수 있도록, 문?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 내주하시는 캐뉼라 Introcan 22G </td><td> 브라운 </td><td> REF 4252098B </td><td></td></tr><tr><td> Dispase, 100 ML (5000 caseinolytic 단위) </td><td> BD Biosciences </td><td> 354235 </td><td> 15 ML 튜브 4 ML에 나누어지는, -20 ° C에서 저장 </td></tr><tr><td> Biozym 플라크 아가로 오스 </td><td> Biozym </td><td> 840101 </td><td> H 2 O 1 % W / V </td></tr><tr><td> 소 췌장, 2000 Kunitz 단위 / 유리 병에서 Deoxyribonuclease I </td><td> 시그마 - 알드리치 </td><td> D4263 </td><td> 갓 300 μl DMEM에 1 약병의 콘텐츠를 분해 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 22320-022 </td><td> 사용 등의 제조업체 (낮은 G에서 제공lucose, Pyruvate, HEPES) </td></tr><tr><td> 셀 스트레이너 (100 μm, 75 μm) </td><td> BD 팔콘 </td><td> 352360, 352350 </td><td></td></tr><tr><td> 나일론 메쉬 (48 μm, 30 μm) </td><td> Bückmann GmbH의 </td><td> 03-48/26-1020, 03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50 μm 필터 </td><td> PARTEC </td><td> 04-0042-2317 </td><td></td></tr><tr><td> 안티 - 마우스 CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td> 클론 93, 정화 </td></tr><tr><td> 안티 - 마우스 F4/80 </td><td> BioLegend </td><td> 123116 </td><td> 클론 BM8, APC 결합 </td></tr><tr><td> 안티 - 마우스 CD11b </td><td> BioLegend </td><td> 101208 </td><td> 클론 M1/70, PE 결합 </td></tr><tr><td> 안티 - 마우스 CD11c </td><td> BioLegend </td><td> 117310 </td><td> 클론 N418, APC 결합 </td></tr><tr><td> 안티 - 마우스CD45 </td><td> BioLegend </td><td> 103102 </td><td> 클론 30 F11, 정화 </td></tr><tr><td> 안티 - 마우스 CD19 </td><td> eBioscience </td><td> 12-0193-83 </td><td> eBio 1D3, PE 결합 </td></tr><tr><td> polyclonal 염소 안티 쥐 IgG </td><td> BD Pharmingen </td><td> 550767 </td><td> polyclonal, PE 결합 </td></tr><tr><td></td><td></td><td></td><td> 대체 fluorochromes에 커플 링 된 항체가 사용할 수있는 흐름 cytometer와 레이저에 따라 사용할 수 있습니다. </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

지난 수년 동안 폐의 면역 규제의 다양한 측면에 대한 폐포 타입 II 상피 세포 (AECII)의 후원금이 점차 인정 받고 있습니다. AECII는 염증 항공의 시토 킨 생산에 참여하고 감염 및 T-세포 매개 autoimmunity 1-8 모두에서 세포를 항원 제시로도 역할을 표시하고 있습니다. 따라서, 그들은 그러한에게기도 하이퍼 반응 등 임상 상황에서도 특별히 흥미있는 외국인뿐만 아니라 자기 항원 직접 또는 간접적 AECII을 타겟팅 감염. 그러나, 건강한 폐뿐만 아니라 염증의 폐포 타입 II 상피 세포에 의해 제공 자세한 immunologic 기능에 대한 우리의 이해는 단편 남아 있습니다. AECII 기능에 관한 많은 연구가 9-12 마우스 또는 인간의 폐포 상피 세포 라인을 사용하여 수행됩니다. 세포 라인과 함께 일하는 것은 확실히 같은 많은 수의 가용성 O와 같은 혜택을 제공합니다광범위한 분석을위한 F 세포. 그러나, 우리는 기본 손쥐 AECII의 사용이 감염이나 면역 염증과 같은 복잡한 과정이 세포 유형의 역할에 대한 이해를 갖게 될 것입니다. 기본 손쥐 AECII들은 분석 설정에서 역할을 모두 추가 외부 요인에 따라되었습니다 즉, 같은 호흡기 질환을 앓고있는 동물에서 직접 분리 할 수​​ 있습니다. 예를 들어, 가능한 AECII은 intranasally 주로 복제 13이 세포를 대상으로 인플루엔자 바이러스에 감염된 생쥐에서 분리 할 수 있습니다. 중요한 것은, 건강한 쥐에서 분리 AECII의 전 생체 감염을 통해 감염에 장착 된 세포 반응의 연구는 더욱 확장 할 수 있습니다. 주요 손쥐 AECII의 절연에 대한 프로토콜은, CD11c, CD11b, F4/80에 대한 특정 항체로 인한 세포 현탁액의 라벨 다음에 마우스 폐의 효소 소화에 기초CD19, CD45 및 CD16/CD32. 세분화 된 AECII 그 다음 높은 라벨과 측면의 분산 (SSC 높은) 세포 인구로 식별되며 3 분류 형광 활성 세포로 구분됩니다. 마우스 폐에서 기본 상피 세포를 분리하는 다른 방법에 비해, 부정적인 선택에 의해 AECII의 흐름 cytometric 절연을위한 프로토콜은 비교적 짧은 시간에 손길이 닿지 않은, 매우 실용적 순수 AECII를 얻을. 또한, 및 패닝과 림프구의 고갈에 의한 절연이 기존의 방법과는 달리 항체 - 커플 링 자석 구슬 14, 15의 바인딩을 통해, 흐름 cytometric 셀 정렬은 셀 크기와 세분화에 의해 차별을 할 수 있습니다. 흐름 cytometric 셀 정렬을위한 장비를 사용할 수 있는지 감안할 때, 설명 절차는 상대적으로 낮은 비용으로 적용 할 수 있습니다. 표준 항체 및 폐 붕괴에 대한 효소 등 자기와 같은 별도의 시약 옆에구슬이 필요합니다. 절연 세포가 체외 문화와 T-세포 자극의 assays에서뿐만 아니라 transcriptome, 프로테옴 또는 secretome 분석 3, 4 등이 포함 기능 및 분자 연구, 다양한 적합합니다.

건강한 쥐에서 분리 폐 세포 현탁액을 정렬하면 AECII 게이트는 일반적으로 42 정도 차지합니다 ± 모든 이벤트의 10 %입니다. 이 비율은 초기 세포 현탁액은기도에 채용 림프구와 기타 면역 세포의 상당히 높은 비율을 포함하므로, 이러한 바이러스 감염과 같은 호흡기 조건에 마우스를 사용하는 경우 현저하게 낮을 수 있습니다. 3 일 다음과 같은 감염에 IAV 감염된 폐 격리 AECII 위해 우리는 약 50 % AEC...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

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

우리는 흐름 cytometric 부정적인 선택하여 기본 손쥐 유형 II 폐포 상피 세포 (AECII)의 급속한 절연을 설명합니다. 이 AECII 높은 생존 능력과 정결을 표시하고 면역 또는 전염성 질환과 같은 호흡기 질환에서의 역할에 대한 기능과 분자 연구 광범위한 적합합니다.

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 ...

flow-cytometric-isolation-primary-murine-type-ii-alveolar-epithelial

Research

JoVE Journal

Immunology and Infection

26.1K 조회수.  Helmholtz Centre for Infection Research. 우리는 흐름 cytometric 부정적인 선택하여 기본 손쥐 유형 II 폐포 상피 세포 (AECII)의 급속한 절연을 설명합니다. 이 AECII 높은 생존 능력과 정결을 표시하고 면역 또는 전염성 질환과 같은 호흡기 질환에서의 역할에 대한 기능과 분자 연구 광범위한 적합합니다.

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

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

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

에 의해 제작 상피 손상의 분석<em&gt; Entamoeba 아메바</em&gt; 감염

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

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 and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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 ...

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 and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...