이 작품은 UNCF - 머크 대학원 과학 연구 논문 장학생 (JLM), 국립 심장, 폐, 및 혈액 연구소 부여 RO1 - HL - 62228 (JSJ)와 RO1 - HL - 073990 (JSJ)에 의해 지원되었다.

1. 행크스 &#39;잔액 소금 솔루션의 준비 (HBSS) <ol><li> 압력솥 1 L의 유리 비커는 1000 ML은 실린더 졸업하고, 바 저어. </li><li> 두 번 증류수로 900 ML 표시에 autoclaved 1 L의 유리 비커에 HBSS 분말 솔루션 Reconstitute. </li><li> Hepes 나 + 소금 3.4 g와 HBSS 용액을 혼합하고 집중 HCL 또는 NaOH을 사용 7.35-7.41 사이의 값을 산도를 조정합니다. </li><li> 원하는 산도가 획득하고 안정적​​인 유지되면 알부민이 완전히 해소되기 전까지 실온에서 솔루션에 알부민 소 혈청 5g을 추가합니다. </li><li> 전체 볼륨 1000 ML 표시에까지 솔루션, antimyotic, 항생제를 두 번 증류수 10 ML을 추가합니다. </li><li> 철저하게 솔루션을 저어. </li><li> HBSS 용액을 필터하고 나중에 사용하기 위해 50 ML 팔콘 튜브에 그것을 구분합니다. 추가 튜브는 냉동과 나중에 사용할 수 있습니다. </li></ol> 2. 동물 PreparatioN - Tracheal 튜브의 삽입 <ol><li> 필요한 악기 및 tracheal 튜브 (즉, 포셉, 가위, tracheal 튜브 3-0 블랙 꼰의 봉합, 작은 동물 환풍기 등) 삽입에 대한 자료와 트레이를 준비합니다. </li><li> 쥐 1 xylazine 2 케타민을의 비율에 케타민을 / xylazine 칵테일과 함께 완전히 anesthetized되면​​, 쥐의 복부, 가슴, 그리고 목구멍을 면도. 깊은 마취 상태가 달성되었는지 확인하려면, 쥐의 발가락을 꼬집어 및 사지의 페달 철회 있는지 확인하십시오. 쥐의 모든 incisions하기 전에 깊은 anesthetized 있는지 확인하십시오. </li><li> 30 ° 각도로 경사하는 도마에 제기 머리와 쥐 장소 </li><li> 중반 복부에서 아래턱 아래로 피부 절개를합니다. 기관을 노출하기 위해 목 앞쪽에 삼각형의 근육을 멀리 해부하다. </li><li> 기관에 따라 3-0 검은 꼰 치료를 놓습니다. </li><li> 연골 고리 사이의 기관에 절개를합니다D tracheal 튜브를 삽입합니다. 기관에 tracheal 튜브를 확보하기 위해 단단히 봉합을 묶어. </li><li> 작은 동물의 호흡에 tracheal 튜브를 연결합니다. </li></ol> 3. 면역 세포의 격리를위한 악기 준비 <ol><li> 면역 세포 격리 절차에 대한 인 스트 루먼트 (즉, 포셉, 가위, HBSS, 바늘 빠른 선배를 통해 소프트 테플론 24 게이지 박스터 intravascular을 다듬은 사람)와 트레이를 준비 </li><li> 이 10cc 주사기를 준비, HBSS가 들어있는 세포를 aspirating위한 pericardial SAC 및 보유, 다른 하나에 버퍼를 소개하는 HBSS 한 주사기를 입력하십시오. 3.2.1. 격리 절차 원래 길이의 1 / 2 4분의 3으로 바늘 빠른 선배 (1.6 ㎝) 이상 24 게이지 박스터 intravascular의 테플론 팁을 줄이십시오. </li><li> 모든 주사기, 버퍼, 그리고 빈 15 ML 팔콘 튜브 (절차가 완료되면 압축을 푼 유체를 잡아) 얼음에 놓으십시오. </li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 4. 면역 세포 격리 <ol><li> 당신은 칼 모양의 프로세스를 도달할 때까지 복부 벽 (linea 알바 따라)에 중간선 절개를합니다. </li><li> 약간 옆으로 칼 모양의 프로세스 시작, 겨드랑이쪽으로 각도로 계속 갈빗대를 통해 양국 incisions합니다. 폐, 심장, 또는 심낭을 구멍하지 않도록주의하십시오. </li><li> 흉골 양쪽에 incisions가 완료되면, 중간에 가로 절단, 낮은 갈비에서 다이어프램 멀리 해부하다. 참고 : 심낭의 다이어프램과 손상을 방지하려면 흉골에 연결하여 어디까지 인식한다. 심낭은 횡경막의 반대편에 연결되어있는 곳을 참고하는 랜드마크로 낫 모양의의 인대를 사용합니다. 심낭을 절단하는 경우 또한, 세포를 분리하는 기능이 손실됩니다. </li><li> 부드럽게 흉강과 마음을 (여전히 심낭에 의해 캡슐) 노출 ribcage 한쪽 리프트.</li><li> HBSS 채워진 주사기를 가지고 흉골과 심장 (선호 더 주동이의 꼬리 이상) 사이의 약 절반 방법 pericardial SAC의 지점을 찾아의 테플론 팁을 삽입 심낭의 두 층을 통해 신속 - 캐서린 단축. </li><li> 카테 테르가 자리에되면, HBBS 버퍼 2 ~ 3 ML과 pericardial 공간을 채우기 부드럽게 진행합니다. 당신은 pericardial 공간이 확장 통보시 카테 테르가 올바르게 위치하면 즉시 알 수 있습니다. 더 충전이 목격되지 않으면 철회하고 빠른 선배를 재조 정할. </li><li> pericardial SAC가 채워진 경우, 다른 10 CC에 주사기를 가지고 심낭에 다시 팁을 장소와 버퍼를 aspirating 시작합니다. 동일한 항목 구멍 사용할 수 있습니다 또는 다른가 만들 수 있습니다. 특히 심장의 아래쪽에 흡입을 제공하면서 끝이 pericardial SAC의 벽에 너무 가까이 오지 않는 있는지 확인하십시오. 이것은 세포가 포함된 세포의 pericardial SAC와 손실을 펑쳐링으로 이어질 수S. </li><li> 버퍼 aspirating 동안 카테 테르 팁은 가능한 한 많은 버퍼를 수집하기 위해 pericardial SAC 내부 주위를 이동할 수 있습니다. </li></ol> 참고 : 이렇게 여러분이 약간 심장막 찢어하고 원래 구멍보다 큰 가슴의 상단에있는 구멍을 만들어 않아도 당황하지 마십시오. 한 심장막 아직 충분히 마음을 주변으로 당신은 여전히​​ 버퍼의 낮은 양의 불구하고 가슴에 또 다른 씻고을 수행할 수 있습니다. 세포의 최적의 금액 수집, 3-4 추가적인 세척이 필요합니다. <ol start="9"><li> 얼음에 놓여 있던 팔콘 튜브로 추출 버퍼를 놓습니다. </li><li> 심장이나 폐, 및 / 또는 추가 연구를 위해 필요한 경우 다른 조직을 제거합니다. 면역 세포 및 기타 조직의 수집 후, 쥐 신속하게 그리고 편안하게 euthanized해야합니다. </li><li> 4 200g에서 10 분 ° C.에 대한 수집 면역 세포 샘플을 원심 분리기 항상 얼음에 튜브를 유지사용하지 않을 때 인치 </li><li> Hyclone 버퍼 또는 HBSS 1 ML에 뜨는와 reconstitute 펠렛을 제거합니다. </li></ol> 참고 : 면역 세포 격리 절차 후 각 마음에서 얻은 면역 세포의 수를 계산하는 것이 중요합니다. 5. 대표 결과 : toluidine 파란색과 safranin O / alcian 파란이 기술과 스테인드로 얻은 고립 심장 마스트 세포의 예제는 각각 2A 및 2B 수치로 표시됩니다. 그림 3, 심장 마스트에서 볼 수 있듯이, 같은 화합물 80분의 48과 모건 외에서 설명한대로 물질 P로 알려진 activators와 치료 후 히스타민을 풀어 자신의 능력을 유지. CD4의 유동세포계측법에서 9 대표 분산형 플롯 + 세포와 CD8 + 세포는 각각 그림 4와 5에 제공됩니다. T - lymphocytes는 더욱 식별이 기술에서 세포 인구로 phenotyped 있습니다. 심장 면역 세포 격리 절차의 그림 1. 플로우 차트. <img alt="그림 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . 심장 마스트 세포의 그림 2 Histological 대표 이미지는 다음과 같이 스테인드 있습니다 : (A) toluidine 청색과 함께 (B) safranin O / alcian 파란색입니다. <img alt="그림 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> 그림 3. epicardial 심근 지역에서 분리된 화합물 80분의 48 (10μg/mL)와 물질 P (10μM)로 자극을 심장 마스트 세포의 기능 연구. <img alt="그림 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> 그림 4. 격리 CD4 + 세포 물질 P (1:100 dilut으로 표시 대표 유동세포계측법의 분산형 플롯이온). <img alt="그림 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> 그림 5. 물질 P (1:100 희석)로 표시 격리 CD8 + 세포의 대표 유동세포계측법.

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우리는 공개 아무것도 없어.

심장 면역 세포를 분리 이러한 방법은 일반적으로 전통적인 효소 소화 절연 기술 중에 발생하는 원치 않는 작동하지 않고 자신의 기능, 표현형, 그리고 성숙의 분석을 수 있습니다. 이 기술을 얻은 세포의 평균 총수는 쥐 심장 당 400,000 주위되며, 이러한 세포의 차동 얼룩 및 유동세포계측법 분석 T - lymphocytes의 혼합 인구 (~ 70 %), macrophages (~ 12 %)와 마스트 세포와를 나타냅니다 이 기법의 사용. (~ 12 ...

<table border="1"><tbody><tr><td> 시약 및 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 행크스 &#39;균형 소금 </td><td> 시그마 </td><td> H4891 </td><td> 한 병은 행크스 &#39;대차 소금 1 패하게 솔루션. Reconstitute 행크스 &#39;밸런스 소금 절차의 일환으로 솔루션입니다. </td></tr><tr><td> 항생제 - Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H3784 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A3912 </td><td></td></tr><tr><td> 250 ML의 Nalogene 필터 병 </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> 피셔 과학 </td><td> CAS 7647-01-0 </td><td> 6 N 들어, deionized에 HCL의 동일한 볼륨을 추가물. </td></tr><tr><td> 10 N NaOH </td><td> 피셔 과학 </td><td> SC267 </td><td> 5 N 들어, 탈이온수에 NaOH 동등한 볼륨을 추가합니다. </td></tr><tr><td> 두 번 증류수 </td><td></td><td></td><td></td></tr></tbody></table> 특정 장비의 테이블 : <table border="1"><tbody><tr><td> 시약 및 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 3 - O 봉합사 실크 꼰 </td><td> Ethicon 제품 </td><td> SA64H </td><td></td></tr><tr><td> 작은 동물 환풍기 </td><td> CWE 법인 </td><td> SAR - 830 </td><td> 분 당 40.5 심호흡으로 호흡 속도를 설정합니다. </td></tr><tr><td> 베벨 18 게이지 바늘 </td><td> BD Biosciences </td><td> 305,199 </td><td> tracheal 튜브로 사용하기 위해 원래 길이의 1 / 2 4분의 3로 바늘을 낸다. </td></tr><tr><td> 24 게이지 박스터 빠른 선배 카테터의 테플론 팁 (바늘이 제거된) </td><td> 벡스터 </td><td> 2N111 </td><td> 1 / 2 4분의 3로 테플론 팁 길이를 줄입니다. </td></tr><tr><td> 10 CC 주사기 </td><td> 피셔 과학 </td><td> 14-823 - 16B </td><td></td></tr><tr><td> 15 ML 팔콘 튜브 </td><td> BD Biosciences </td><td> 352,097 </td><td></td></tr><tr><td> Sorvall RT6000B 냉장 원심 분리기 </td><td> 피셔 과학 </td><td> 75-004-377 </td><td> 사전 냉각 기억하고 4 유지 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemocytometer </td><td> Hausser 과학 </td><td> HS - LB - 1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

심장 면역 세포들은 많은 심장 질환의 병리 리모델링에서 재생 역할에 대한 관심을 쫒아오고 있습니다 마스트 세포, T - 세포 및 macrophages 포함 1-5 이들 면역 세포;. 저장하고 크린 시토킨 및 포함하여 생물학적으로 활성 중재자 다양한 릴리스 같은 tryptase 같은 프로 테아제. 6-8이 중재자는 매트릭스 metalloproteinases를 활성화하거나 modulating 심장 섬유아 세포 &#39;기능에 의해 콜라겐 축적을 유발하여 세포외 기질 대사의 주요 선수로 표시되었습니다. 9-11하지만, 심장 면역 세포를 분리에 사용할 기술이있다 그들은 심근 조직을 소화 박테리아 collagenase을 사용하기 때문에 문제가되었습니다. 이 기술은 면역 세포의 활성화 및 따라서 기능의 손실을 초래합니다. 예를 들어, 심장 마스트 세포가 degranulation의 원인이 화합물에 훨씬 적은 반응이된다. 12 따라서, 우리는 t을 허용하는 기술을 개발그는 심장 질환에서 이러한 세포의 역할에 대한 이해로 이어질 것입니다 기능적 심장 면역 세포의 분리. 13, 14 이 방법은 심장의 해부 쥐의 검상 돌기 프로세스, 겨드랑이와 낫 모양의 제품 인대의 위치, 그리고 심낭과 친숙이 필요합니다. 이러한 랜드마크 프로 시저의 성공을 증가하고 심장 면역 세포의 높은 수율을 보장하기 위해 중요합니다. 이러한 격리 심장 면역 세포는 그 기능 표현형, 만기, 그리고 다른 심장 세포와 공동 배양 실험들은 상호 작용의 더 나은 이해를 얻을 수의 특성에 사용될 수 있습니다.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

마음에서 기능적 면역 세포를 분리를위한이 방법은 이러한 세포의 감소 반응의 결과로, 원치 않는 면역 세포 활성화의 원인이 collagenase의 소화의 종래의 방법에 대한 대안을 제공합니다. 격리 우리의 방법은 효소 소화와 관련된 문제를 피함으로써 기능 심장병 면역 세포를 산출.

심장 기능 면역 세포의 분리

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K 조회수.  University of South Carolina- School of Medicine. 마음에서 기능적 면역 세포를 분리를위한이 방법은 이러한 세포의 감소 반응의 결과로, 원치 않는 면역 세포 활성화의 원인이 collagenase의 소화의 종래의 방법에 대한 대안을 제공합니다. 격리 우리의 방법은 효소 소화와 관련된 문제를 피함으로써 기능 심장병 면역 세포를 산출.

비디오: Isolation of Functional Cardiac Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

셀을 별 모양으로 일곱 단계

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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

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

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

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

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

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

지방 조직 면역 세포의 분리

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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, Characterization and Functional Examination of the Gingival Immune Cell Network

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

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

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

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

Isolation 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 ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

복 막 파생 돛대 세포 및 그들의 기능 특성화 캘리포니아2 +의 격리-이미징 및 Degranulation 분석 실험

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...