저자 microphotographs 그의 도움을 셀 정렬과 피터 Mottram 함께 그녀의 도움 LSU의 유동세포계측법 코어 시설에서 마릴린 디트리히에 감사드립니다. 이 작품은 비행 수행자 의학 연구소, LSU 경쟁력 연구 프로그램 수상, 그리고 NIH / NIAID 기금 P20 RR020159 및 R03AI081171에 의해 후원되었다.

1. 폐 재관류 및 단일 세포 현탁액 <ol><li> 대퇴 정맥을 통해 intraperitoneal 케타민을 / xylazine 마취 혼합물의 주입 (MG / 마우스 케타민을 1.8, xylazine 0.19) 및 과다와 마우스를 안락사 </li><li> 절단하고 부드럽게 복막의 바깥쪽 피부를 다시 당겨 흉강을 폭로. 심장과 폐 모두 노출 갈빗대 절단하여 다이어프램을 열 진행합니다. </li><li> EDTA (에틸렌 다이아 민 테트라 초산) - HBSS (칼슘 / 마그네슘없는 HBSS에 1mM EDTA (에틸렌 다이아 민 테트라 초산))로 가득 5 ML의 주사기를 사용하여 부드럽게 폐가 Perfuse. 25 G 바늘을 연결하고 우심실에 바늘을 삽입합니다. 주사하는 동안 누출을 방지하기 위해, 집게를 사용하여 바늘 주위 myocardiac 조직을 확보. 상수 압력을 유지하면서 부드럽게 솔루션을 주입. 정확한 재관류는 폐암의 인플레이션과 분홍색 / 흰색 색상의 변화가 발생합니다. </li><li> 이 조직에서 잠재적인 오염을 폐기하기 위해 림프절을 배수 제거합니다. </li><li> 수집폐 조직은 얼음에 페트리 접시에 전송하고, 면도날을 사용하여 작은 조각으로 썰어. </li><li> 5 ML / collagenase의 소화 솔루션의 폐를 포함하는 gentleMACS C 관에 근거 조직을 전송 (collagenase 유형 1A 0.5 MG / ML 플러스 유형 IV 소 췌장 DNase 20 5% FBS, 100 U / ML 페니실린과 100 μg이 포함된 HBSS에 μg / ML ML의 스트렙토 마이신) /. </li><li> 단일 세포 현탁액을 얻기 위해 gentleMACS의 dissociator을 사용합니다. m_lung_01 : 프로그램을 선택합니다. 30 분 37 ° C에서 샘플을 품어. 관에게 조직 조각을 resuspend 위해 5 분마다 흔들어. 프로그램 m_lung_02 진행합니다. </li><li> 4 335 X g에서 10 분 동안 15 ML 원뿔 튜브와 원심 분리기 샘플을 전송 ° C. 이 단계 후, 4 시에 시약 및 centrifugations을 유지 ° C가 세포 생존의 감소를 방지합니다. </li><li> 뜨는을 취소하고 실온에서 1 분 ACK lysing 버퍼 2 ML을 추가하여 나머지 적혈구를 lyse. 13 세포를 씻으십시오4 335 X g에서 10 분 동안 차가운 PBS/0.5 %의 BSA와 원심 분리기 ° C.의 ML </li><li> 차가운 PBS/0.5 %의 BSA 5 ML에 뜨는, resuspend 총 세포를 버리고 100 μm의 나일론 메쉬를 철저하게 전달합니다. </li><li> trypan 파랑 제외 염료를 사용하여 전체 휴대폰 번호를 확인합니다. </li></ol> 2. 자기 고립과 CD11c + 세포의 농축 <ol><li> 4 ° C에서 200 X g에서 10 분간 단일 세포 현탁액을 원심 분리기와 뜨는 폐기. </li><li> 10 8 전체 세포마다 분리 버퍼 400 μl에 Resuspend 세포 펠렛 (PBS, 0.5 % BSA, 2 MM EDTA (에틸렌 다이아 민 테트라 초산)). 4 30 분 anti-CD16/CD32 항체 (0.5μg / 10 6 셀)를 사용하여 바인딩을 차단 FC - 중재 unspecific 항체 ° C. </li><li> 4 200 X g에서 10 분 동안 차가운 분리 버퍼와 원심 분리기 10 ML과 세포를 씻으 ° C. 분리 버퍼 400 μl의 세포를 Resuspend. </li><li> 10 8 회 CD11c MicroBeads 100 μl 추가 </&gt; 전체 세포를 먹어 볼래요. 잘 섞어 냉장고에 15 분 (2-8 ° C)를 품어. </li><li> 4 ° C에서 200 X g에서 10 분 동안 차가운 분리 버퍼와 원심 분리기 10 ML과 세포를 씻고 뜨는 폐기. </li><li> 감기 분리 버퍼 3 ML에있는 세포 펠렛을 Resuspend. </li><li> 프로그램 posseld를 선택하여 autoMACS 프로 TM 구분 기호를 사용하여 자기 분리를 수행합니다. 긍정적인 분율 (0.5 ML)를 수집합니다. </li></ol> 3. 기존 돌기 세포 (CDC) 격리 <ol><li> 반 CD11c PE - Cy7 및 anti-IA/IE (MHC - II) FITC 항체 30 분 동안 4 ° C.와 함께 품어 풍부한 CD11c 양성 세포 현탁액 항체의 최적화된 농도는 세포의 0.5 μg / 10 6입니다. </li><li> 4 335 X g에서 10 분 동안 차가운 PBS/0.5 %의 BSA와 원심 분리기 ° C.로 세포를 씻어 Resuspend PBS/0.5 %의 BSA의 0.5 ML의 세포와 40 μm의 나일론 메쉬을 통해 전달합니다. </li><li> 로 이동순결 모드에서 FACS의 아리아 셀 분류기를 사용하여 세포를 정렬합니다. 정화 세포의 손상을 방지하기 위해 수집 튜브에 PBS/0.5 %의 BSA 100 μl를 추가합니다. 셀 정렬 과정에 걸쳐 냉장 온도에서 세포 보관하십시오. </li></ol> 4. 단일 세포 현탁액 및 CD11c + 세포 농축을 얻기위한 대체 프로토콜 <ol><li> gentleMACS의 dissociator의 교체에서 단계 1.5에서 접지된 폐 조직은 37 ° C.에서 1 시간 동안 폐암과 부화 당 collagenase 솔루션의 5 ML을 포함한 15 ML 원뿔 튜브로 전송할 수 있습니다 와동은 세포 매 15 분 조직 조각을 resuspend하기 위해서. 에서 밖으로 20 G 바늘에 연결된 3 ML의 주사기를 통해 샘플에게 6-8 번 통과하여 조직을 중단. 과정에서 거품을 만들어 피하십시오, 달리 세포 생존이 위험해질 것입니다. 단일 세포 현탁액이 얻어되면 1.8 단계로 진행합니다. </li><li> AutoMACSPro TM separa의 교체에토르, CD11c 세포의 강화를위한 자기 절연은 또한 두 맥 컬럼을 통해 수동으로 세포 현탁액을 전달하여 수행할 수 있습니다. 해당 컬럼의 선택은 샘플 크기에 따라 달라집니다. </li></ol> 5. 대표 결과 : 폐 CDC는 CD11c 하이 / MHC - II 안녕하세요 셀룰라 인구로 구분됩니다. 마찬가지로 그림에 표시됩니다. 1, CDC는 비장 (4.8 %) 등의 다른 조직에 비해 1.4 %의 적은 비율을 나타냅니다. 그러나, 비장과는 달리, 폐 CD11c 양성 세포는 CDC보다 다른 세포 인구를 포함하여, MHC - II의 다른 양의 표현한다. 단일 셀 준비 후, 총 폐 세포의 세포 수율은 3.8-3.0에 대해 조금있는에서 1 % 이상 60-70%의 가능성과 함께 X 10 7 세포 / 폐암은 CDC의 하위 집합을 표현했다. 이 비율은 전체 폐암 CDC가 약 10 T를 증가했던 CD11c 자성 분리 후 증가되었습니다원래 준비 (그림 2A)에서 imes (&gt; 16 %). 그러나, MHC - II (CD11c 하이 / MHC - II 이오, macrophages)의 낮은 금액을 표현하는 CD11c 세포가 폐 CDC 집합과 혼합되었다 높은 오염 세포 인구 (&gt; 70 %) 대표. 마찬가지로 그림에 표시됩니다. 2B, 그 세포는 CDC 순도가&gt; 96 %에 도달 셀 정렬 단계 이후에 제거했다. 전체 절차 이후에 CDC의 일반적인 수율은 5 X 10 4 셀 / 폐되었습니다. Microphotographs은 전형적인 dendrites (상단 패널)와 둥근 세포로 CDC의 형태학의 특성을 보여줍니다. 한편, CD11c 안녕하세요 / MHC - II 소호 macrophages (MΦ, 낮은 패널) 12 전형적인 세포 protuberances를 보여줍니다 morphologically 다른 하위 집합을 나타낸다. <img alt="그림 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> 마우스 폐와 비장 그림 1. 차동 DC 주파수. 폐C57BL / 6 마​​우스에서 차 비장 조직을 collagenase와 수집 치료를했다. collagenase의 소화 다음, 세포는 반 마우스 CD11c - PE - Cy7 및 안티 마우스 IA / IE - FITC로 얼룩진했다. 대표 유동세포계측법의 플롯은 폐와 비장의 CDC의 비율 (CD11c 하이 / MHC - II 안녕)를 보여줍니다. <img alt="그림 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> 그림 2. 마우스 폐에서 CDC의 분리. 마우스 폐 단일 세포 현탁액을 방지 CD11c microbeads으로 표시하고 자동으로 셀 분리 통과.) 대표 줄거리는 CDC의 비율 (CD11c 하이 / MHC - II 안녕)를 보여 주며되었습니다 Macrophages (MΦ, CD11c 안녕하세요 / MHC - II 싸다) CD11c 농축 후 인구. 농축 분율의 추가 얼룩 방지 - 마우스 CD11c - PE - Cy7 및 안티 마우스 IA / IE - FITC을 사용하여 따라갔다. 이중 긍정 세포 분류와 유동세포계측법에 의해 분석되었다. 에자신의 형태를 확인, 세포가 수정 라이트 ​​- Giemsa의 얼룩과 cytospun과 스테인드되었습니다. B) 대표 플롯은 CDC의 비율과 셀 정렬 후 MΦ을 보여줍니다. Microphotographs는 폐 질병 통제 센터와 폐 MΦ의 대표 이미지를 보여줍니다. 스케일 바 = 20 μm의.

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저자는 공개 아무것도 없어.

폐 마우스 DC의 격리가 호흡기 자극의 다양한 연구를위한 중요한 기술이다. 이러한 세포를 얻는 과정은 세포의 손실뿐만 아니라 세포 생존과 정결을 방지 중요한 단계를 포함하고 있습니다. 수령 전에 폐가 Perfusing 것은 물론 오염 물질을 감소 적혈구와 같은 주변 세포를 제거하는 데 도움이 될 것입니다. 폐 다수의 처리 때 자동 분리의 사용 advanteogus 수있다 그렇지 않으면 수동으로 완료되면 collag...

<table width="200" border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> ACK lysing 버퍼 </td><td> Invitrogen </td><td> D6 - 0005DG </td><td></td></tr><tr><td> 안티 마우스 CD11c (HL3) </td><td> BD Pharmingen </td><td> 5,580,979 </td><td> PE - Cy7 복합 </td></tr><tr><td> 안티 마우스 IA / IE (269) </td><td> BD - Pharmingen </td><td> 553,623 </td><td> 복합 FITC </td></tr><tr><td> Collangenase 입력 1A </td><td> 시그마 </td><td> 9891 - 500MG </td><td></td></tr><tr><td> 세포 strainers </td><td> BD 팔콘 </td><td> 352340, 352360 </td><td></td></tr><tr><td> CD11c (N418) Microbeads </td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNase I </td><td> 시그마 </td><td> D5025 - 150KU </td><td></td></tr><tr><td> 행크의 균형 소금 솔루션 </td><td> Invitrogen </td><td> 14,170 </td><td></td></tr><tr><td> Hepes 버퍼 솔루션 </td><td> Invitrogen </td><td> 15,630 </td><td></td></tr><tr><td> 배양 접시 60mm </td><td> BD 팔콘 </td><td> 351,016 </td><td></td></tr><tr><td> GentleMACS ™ C 튜브 </td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> 젠틀 맥에서의 dissociator </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS - PRO ™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASCS 아리아 </td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

폐암 돌기 세포 (DC)는 호흡기 tolerogenic 응답의 제어뿐만 아니라 3 침입 병원균 1,2를 감지의 근본적인 역할을한다. 폐암 돌기 세포 중 적어도 세 가지 주요 하위 집합은 생쥐에서 설명되었습니다 : 종래의 DC (CDC) 4, plasmacytoid DC (PDC) 5 IFN 생산 킬러 DC (IKDC) 6,7. CDC 집합은 폐 8 가장 유명한 DC의 하위 집합입니다. DC의 하위 집합을 식별하는 것으로 알려진 일반적인 마커는 CD11c, 유형 I 또한, monocytes, macrophages, neutrophils 및 일부 B 세포 9 표현 transmembrane 인테 그린 (β2)입니다. 일부 조직에서는, 마우스 DC를 식별하는 마커로 CD11c를 사용하면 대부분의 CD11c + 세포가 주요 histocompatibility 복잡한 클래스 II (MHC - II)의 높은 수준을 표현 CDC의 하위 집합을 나타냅니다 비장에 같은 유효합니다. 그러나, 폐는 더 이질적인 조직입니다사이드 DC의 하위 집합은 MHC - II의 낮은 수준의 한판 승부 CD11c 높은 수준을 표현 뚜렷한 세포 인구의 높은 비율 있습니다. 의 특성과 대부분 F4/80, 지라 대식 세포 표지 자사의 표현에 따르면, CD11c 하이 MHC - II 그런 건 폐 전지 인구가 잠재적인 DC 전구체 11로, 폐동맥 macrophages 최근 10으로 확인되었습니다. 마우스 PDC 대조적으로, 폐의 면역 반응에 CDC의 특정 역할의 연구는 이러한 세포의 격리에 도움이 될 특정 마커의 부족으로 인해 제한되었습니다. 따라서,이 작품에서 우리는 고도로 정제된 마우스 폐 질병을 분리하는 절차를 설명합니다. 폐 DC의 하위 집합의 분리는 호흡기 병원균뿐만 아니라 폐의 호스트 면역 반응을 일으킬 수있다 환경 요인에 대한 응답에서 이러한 세포의 기능에 대한 통찰력을 얻기 위해 매우 유용한 도구를 나타냅니다.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

마우스 폐 돌기 세포의 고도 정화 준비가 설명되어 있습니다. 구체적인 중점이 종래의 돌기 세포 집합의 고립에 제공됩니다.

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 ...

Research

JoVE Journal

Immunology and Infection

28.9K 조회수.  Louisiana State University. 마우스 폐 돌기 세포의 고도 정화 준비가 설명되어 있습니다. 구체적인 중점이 종래의 돌기 세포 집합의 고립에 제공됩니다.

비디오: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

마우스 복막 구멍 세포의 분리

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

연구

면역학 및 감염병학

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

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

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 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 ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

마우스 수지상 세포 하위 집합의 분리를위한 효율적이고 높은 수율 방법

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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. ...

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 ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

레지오넬라 뉴모 필라 외부 막 소포 : 분리 및 대 식세포에 그들의 프로 - 염증 가능성 분석

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

면역 및 유동 세포 계측법을 사용하여 기관지 생검의 공간 분포 및 표현형 확인 : 인간의 폐 수지상 세포

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Phenotypical 및 기능 연구에 대 한 인간의 여성 생식 관에서 수지상 세포의 고립

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...