이 작품은 메이요 클리닉 (CLH)에서 조기 경력 개발 보너스로, NINDS (CLH)에서 부여 NS64571 지원하고, 도널드와 프랜시스 Herdrich (CLH)에서 후한 선물로.했습니다 우리는 도움을 메이오 클리닉 유동세포계측법 코어를 감사하고 싶습니다.

1. Intracranial 바이러스 주입 : 다음 기술은 수정 및 실험실 및 동료에 의해 광범위하게 활용되었습니다. 간단히, Theiler의 murine encephalomyelitis 바이러스 (TMEV) 또는 사기 - 감염의 다니엘의 변형의 intracranial 주입 (1, 10) 뇌 침투 leukocytes (BILs)를 이끌어내는 젊은 생쥐 (세 선호 5-6주)에 수행됩니다. 결과는 다니엘의 변형, 콩 변형 및 GDVII 스트​​레인 다를됩니다. BILs을 수확하기위한 목적으로, 생쥐는 TMEV의 2x10 5 PFU을 받고 24 시간 동안 감염됩니다. <ol><li> 자동 1 ML 해밀턴 주사기로 (27 게이지, ¼ 켄달에) 주입 바늘을 첨부 및 바이러스 10 μl를 제공하기 위해 설정할 수 있습니다. </li><li> 공기 방울에 세심한주의와 주사기에 10 μL DMEM에 TMEV을 그립니다. 경우 적절한, 가짜 감염된 생쥐는 바이러스가없는 DMEM 10 μl를 받게됩니다. </li><li> 직전 주사로, 아래 면화 기판과 전선 격자를 포함하는 벨 항아리에 isoflurane의 약 1 ML 잔. </li><li> 발가락 - 핀치에 무심하고 흡입 마취에 의해 고정화 벨 병 속에 와이어 그리드에 자리 마우스 direclty들이 가볍게 anesthetized 될 때까지, 약 10~15초. 마우스는 물질이 자극 및 부식성 수 있듯이, isoflurane 직접 접촉해서는 안됩니다. </li><li> 마취 동안 빠르게 면도 및 inking을 사용하여 두개골에있는 전두엽 피질 영역에 해당하는 좌표의 위치를​​하여 사출에 대한 생쥐를 준비합니다. 주사에 대한 일반적인 위치는 bregma에 앞쪽에 1mm과 오른쪽에있는 화살 봉합 (그림 1) 가로 1mm입니다. stereotactic 사출 일부 실험 상황에 맞게 수 있지만, 우리는 면도 또는 inking없이 무료로 직접 분사이 실험의 대부분에 적합한 것으로 나타났습니다. 두 축의 따라 바늘 지점과 모피 파팅 것은 사출에 적합합니다. </li><li> 주사를 만들기 위해선, 동양 약 3mm 깊이로 뼈를 통해 두개골과 언론에 바늘 직각. </li><li> 해밀턴 주사기 버튼을 눌러 철수하기 전에 두뇌를 입력 바이러스 2 초 대기하여 두뇌로 특급 바이러스. </li><li> 조심스럽게 바늘을 제거하고 깨끗한, 마른 새장에 마우스를 놓으십시오. 마우스 신속하게 마취에서 각성과 분 이내에 정상 기능을 재개하십시오. 동물의 안락사로, 같은 주사 사이트에서 과도한 출혈로 이상 증상을 전시 마우스에 적절한 관심을주고하는 것은 적절한 수 있습니다. 건강과 기관 애니멀 케어 및 사용위원회 지침 국립 연구소에 6 준수를 취급 적절한 동물을 따라야합니다. </li></ol> 2. 브레인 - 침투 백혈구 세포 준비 : <ol><li> 10 ML RPMI 1640, 9 ML Percoll 1 ML 10X PBS 및 튜브 두뇌 제거 프로세스 동안 벤치에서 RT에 앉아 있어야 30 ML의 용량을 저장할 수있는 왕복 하단의 오크 리지 원심 분리기 튜브를 준비합니다. </li><li> 실온 (RT)에 Ficoll - Paque 플러스 가져와. </li><li> 수정 isoflurane 과다 5 동물을 안락사 빠르게 얼음 5 ML RPMI를 포함한 15 ML 원뿔 튜브에 스테인레스 스틸 조직 주걱과 장소를 사용하여 두뇌를 제거합니다. 어떤 상황에서, PBS - 재관류는 BILs 준비에서 말초 혈액 세포를 순환 삭제하기위한 적절한 수 있습니다. 마우스 완전히 정상적으로 anesthetized 사전 PBS - 재관류하는 것입니다. </li><li> 두뇌와 7 ML 유리 Pyrex 브랜드 Tenbroeck 조직 분쇄기로 RPMI 솔루션을 전송하고 부드럽게 10-15 스트로크와 균질. </li><li> 균 질기와 두 pipet 위아래로 Pipet 추가 5 ML RPMI합니다. 전송 뇌 homogenate (약 10 ML) 이전에 준비 왕복 아래 튜브와 부드럽게 반전을 2-3 배 혼합합니다. </li><li> 베크맨 알레 그라 X - 22R 임상 원심 분리기에서 F0360 고정 각도 로터의 RT 30 분 7,800그램 아베의 뇌 homogenate를 스핀. </li><li> 원심 분리 후, 그라디언트의 꼭대기에 떠오르있다 myelin의 잔해를 제거합니다. 붉은 혈액 세포 펠릿 (그림 2) 위의 수레 백혈구 레이어를 수집합니다. </li><li> 50 ML 원뿔로 40 μm의 셀 스트레이너를 통해 백혈구 레이어를 스트레인. RMPI과 함께 50 ML 볼륨에 솔루션을 가져 오십시오. </li><li> 스핀 튜브는 RT에서 5 분 1,500 RPM (600g 아베)에서 임상 원심 분리기에 희석 세포 현탁액을 포함. </li><li> 뜨는을 기음과 세포 펠릿를 수집합니다. 1 ML 외과 버퍼의 펠릿 (1 % 소 혈청 알부민, 0.02 % 나트륨 칼슘 마그네슘 azide와 무료 PBS)를 Resuspend 5 ML 라운드 바텀 외과 튜브에 세포 현탁액을 전송할 수 있습니다. </li><li> 1 ML Ficoll - Paque 플러스와 함께 조심스럽게 밑받침 서스펜션. </li><li> 없이 브레이크와 RT 25 분 임상 원심 분리기에서 2,500 RPM (1400 준)에서 튜브를 스핀. </li><li> 1 ML의 피펫과 새로운 5 ML 외과 튜브로 전달 인터페이스 (그림 2)에서 하얀 솜털 레이어를 제거합니다. 와시 C4 ML 외과 버퍼와 엘스. </li><li> 펠렛 세포로 임상 원심 분리기에서 RT에서 5 분 1,500 RPM (600g 아베)에서 튜브를 스핀. </li><li> 뜨는을 기음과 세포 펠릿를 수집합니다. 적절한 버퍼에 백혈구 펠렛을 Resuspend하고 새로운 외과 튜브에 얼음 계속. </li><li> Trypan 파랑 배제함으로써 세포 생존을 평가하고 유동세포계측법에 의해 분석, 세포를 계산합니다. 일반적으로, 조사는 약 5 × 10 5 세포 / 동물를 얻기 기대할 수 있습니다. </li></ol> 3. 흐름 cytometric immunophenotyping <ol><li> leukocytes을 분리하고 계산 후, 임상 원심 분리기에서 1,500 RPM (600 준)에서 3 분 동안 세포를 스핀. </li><li> 다음 사항을 포함하는 버퍼를 차단에서 펠렛을 Resuspend : 2.4G2 하이 브리 도마 (FC 차단, anti-CD16/32)에서 뜨는 외과 버퍼, 그리고 10시 5분 1초의 비율로 태아 소 혈청과 4 30 분 품어 ° C . </li><li> 준비하고 4 30 분 1:200과 부화의 농도에서 차단된 세포 ° 어둠 속에 C로 세포 항원에 대한 항체 복합을 추가합니다. 스테인과 96 잘 V - 하단 플레이트에 200 μl의 세포를 품어. 적절한 컨트롤이 필요한 흠없는 셀 및 fluorochrome 보상 샘플을 포함합니다. </li><li> 96 - 웰 플레이트에 대한 적절한 로터를 사용하여 임상 원심 분리기에서 1,500 RPM (600 준) 3 분 RT에서 스테인드 세포를 스핀. </li><li> 뜨는을 대기음 부드럽게 3 번 아래 pipetting과로 200 μl 외과 버퍼로 세포를 씻어. </li><li> 두 번 이상 세척 기법을 반복합니다. </li><li> 세척 후, 2 % paraformaldehyde와 외과 튜브로 전송에서 세포를 resuspend. 고정은 Biosafety 레벨 2 시약에 감염된 세포의 흐름 cytometric 분석을 위해 필요합니다. </li><li> 수정된 유동세포계측법 방법 8 다음 BD 외과 Calibur에 고정 셀을 실행하고 오프라인 FlowJo, WinMDI 또는 기타 가능한 유동세포계측법 분석 소프트웨어를 사용하여 파일을 분석할 수 있습니다. </li><li> 샘플 당 50-100,000 이벤트의 분석은 일반적으로 적절한 immunophenotyping 필요합니다. </li></ol> 본 실험에 사용되는 직접 복합 항체 : CD45는 클론 30 F11과 함께 발견되었다. Ly6C / G는 복제 Gr1, RB6 - 8C5과 함께 발견되었다. CD11b는 클론 M1/70과 함께 발견되었다. F4/80는 클론 BM8과 함께 발견되었다. 4. 대표 결과 : 우리는 24 시간 이후 감염 (그림 3)에서 마우스 BILs에 대한 세포 phenotyping 결과를 보여줍니다. Leukocytes이 면역 세포를 감지하는 PerCP - 복합 방지 마우스 CD45 물들일 있었 PE - 복합 백신 마우스 Ly6C / G 세포를 감지하는 염증성 monocytes, APC - 복합 백신 마우스 CD11b와 APC - 복합 백신 마우스 F4/80를 검색하는 단핵구 혈통니다. 분석 BD 외과 Calibur 악기와 함께 연주했다. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="그림 1"/> 그림 1. 사출 사이트의 해부 지방화. 사출 사이트 bregma에 앞쪽에 1mm과 오른쪽에있는 화살 치료 측면 1mm에 위치해 있습니다. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="그림 2"/> 그림 2. leukocytes 및 BILs의 기울기 분리 일러스트. Leukocytes 처음에는 직접 myelin의 파편 층 아래와 Percoll 기울기에 RBC 펠렛 위의 수집하고 있습니다. 인터페이스에 하얀 솜털 층 (BILs)은 Ficoll 기울기로부터 수집됩니다. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="그림 3"/> 그림 3. 24 시간 후 감염. 마우스 leukocytes에서 두뇌 infilatrating leukocytes의 Immunophenotype은 조직 intracranial 감염 후 24 시간에서 동물에서 준비 homogenates의 차동 밀도 원심 분리에 의해 두뇌에서 고립되었다. 세포는 마우스 CD45에 대한 찬란 - 복합 항체 물들일 있었 Ly6C / G, CD11b, 그리고 F4/80. 초기 게이팅는 앞으로 분산형 (FSC), 셀 크기 (C)의 지표에 대한 CD45, 면역 세포의 마커를하려 의해 수행되었다. CD45 안녕 (A, D), CD45 이오 (B, E) 및 CD45 neg (F, G) 인구는 다음 단핵구와 대식 세포 마커 Ly6C / G, F4/80 (A, B의 상대적인 표현 수준을 분석했다 , F) 및 CD11b (D, E, G). Ly6C / G + F4/80 + CD11b + 염증 monocytes가 주변에서 이러한 세포의 침투와 일치 CD45 안녕 인구 (A, D)에 거의 독점적으로 발견되었습니다. 대조적으로, Ly6C/G-F4/80 이오 CD11b + macrophages은 CD45 이오 (B, E)와 CD45 neg (G) 주민 표현형과 일치 집단에 거의 독점적으로 발견되었습니다. 수많은 실험에서, 우리는 감염되지 않은 또는 사기 - infec의 두뇌에서 CD45 안녕 셀 또는 Ly6C / G + F4/80 + CD11b + 염증 monocytes을 관찰 적이 없다테드 마우스 (데이터가 표시되지 않음).

<ol>
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관심 없음 충돌 선언하지 않습니다.

우리는 정기적으로 뇌 세포 침투 준비의 품질 모두를 결정하는 유동세포계측법를 사용하고, 면역 세포, 2, 9의 다른 인구를 구별합니다. 급성 시간 지점에서 우리 BILs 메서드는 CD45hi 인구뿐만 아니라 CD45lo 인구의 macrophages 높은 비율 이내에 염증 monocytes의 높은 비율을 산출. 이것은 두뇌 속에 재현할 면역 반응이 견고하게 우리의 방법을 특징으로 할 수 없음을 나타냅니다. <p class="jove_con...

<table border="1"><tbody><tr><th> 시약 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> TMEV </td><td> 하우 랩 </td><td> N / A </td><td></td></tr><tr><td> 다니엘의 변형 </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> isoflurane </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> 둥근 바닥 오크 리지 원심 분리기 튜브 </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE 헬스케어 </td><td> 17-0891-02 </td><td></td></tr><tr><td> 10X PBS </td><td> 로체 </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll - Paque 플러스 </td><td> GE 헬스케어 </td><td> 17-1440-02 </td><td></td></tr><tr><td> Trypan 블루 0.4 % (W / V) </td><td> Mediatech </td><td> 25-900 - CI </td><td></td></tr><tr><td> 15 ML 원뿔 튜브 </td><td> BD 팰컨 </td><td> 352097 </td><td></td></tr><tr><td> 7 ML 유리 Pyrex 브랜드 Tenbroeck 조직 연삭기 </td><td> 어부 </td><td> 08-414 - 10B </td><td></td></tr><tr><td> 40 μm의 셀 스트레이너 </td><td> BD 팰컨 </td><td> 352340 </td><td></td></tr><tr><td> 50 ML 원뿔 </td><td> BD 팰컨 </td><td> 352070 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A9647 </td><td></td></tr><tr><td> 나트륨 azide </td><td> 시그마 </td><td> S8032 </td><td></td></tr><tr><td> CMF - PBS </td><td> Mediatech </td><td> 21-040 - CV </td><td></td></tr><tr><td> 외과 튜브 </td><td> BD 팰컨 </td><td> 352054 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 시그마 </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 하이 브리 도마 </td><td> ATCC </td><td> HB - 197 </td><td></td></tr><tr><td> 코스타 V - 바닥 플레이트 </td><td> 코닝 </td><td> 3894 </td><td></td></tr><tr><td> 알레 그라 X - 22R 원심 분리기 또는 동급 </td><td> 베크맨 </td><td> N / A </td><td></td></tr><tr><td> 96 - 웰 플레이트 양동이와 로터 </td><td> 베크맨 </td><td> S2096 </td><td></td></tr><tr><td> 고정 앵글 로터 </td><td> 베크맨 </td><td> F0360 </td><td></td></tr><tr><td> paraformaldehyde </td><td> 시그마 </td><td> P6148 </td><td></td></tr><tr><td> BD 외과 Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> FlowJo 7.5 </td><td> 트리 스타 (주) </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> 클론 : 30 - F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> 클론 : RB6 - 8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> 클론 : M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> 클론 : BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

우리는 생쥐의 두뇌 침투 leukocytes (BILs)를 준비하는 방법을 설명합니다. 우리는 어떻게 뇌 전체에서 침투 세포 백혈구 - 풍부한 인구를 정​​화하기 위해 신속한 intracranial 분사 기술과를 통해 Theiler의 murine encephalomyelitis 바이러스 (TMEV)와 마우스를 감염하는 방법을 보여줍니다. 간단히 마우스는 닫힌 챔버에서 isoflurane과 anesthetized되며 전두엽 피질에 해밀턴 주사기로 주입 자유 손으로입니다. 생쥐는 다음 Tenbroeck 조직 분쇄기로 RPMI에서 추출하여 균질 아르 isoflurane 과다 복용과 전체 뇌 감염 후 여러 시대에서 살해되었습니다. 두뇌 homogenates은 myelin 및 기타 세포 파편을 제거하는 연속 30 % Percoll 기울기를 통해 centrifuged 수 있습니다. 세포 현탁액은 다음 40 μm의에서 긴장 세탁 및 leukocytes을 선택하고 정화하는 불연속 Ficoll - Paque 플러스 기울기에 centrifuged 있습니다. leukocytes 그런 다음 세탁 및 유동세포계측법에 의해 immunophenotyping에 적합한 버퍼에 resuspended 있습니다. 유동세포계측법가 C57BL / 6 생쥐에 감염의 초기 단계에 타고난 면역 세포의 인구를 보여줍니다. 24 시간 이후 감염에서 면역 세포의 여러 하위 집합은 Gr1 +, CD11b +와 F4/80 + 세포의 풍부한 인구와 BILs에 존재한다. 따라서,이 방법은 두뇌의 급성 감염에 대한 면역 반응을 특성화에 유용합니다.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

murine 두뇌에서 침투 leukocytes을 얻는 빠른 방법을 설명합니다. 이 방법은 연속 Percoll 기울기와 백혈구 - 농축 레이어를 선택하고 정화 불연속 Ficoll 기울기를 사용합니다. 절연 leukocytes 그때 흐름 cytometric 측정에 의해 특징 수 있습니다.

브레인 - 침투 Leukocytes의 분리

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K 조회수.  Mayo Clinic College of Medicine. murine 두뇌에서 침투 leukocytes을 얻는 빠른 방법을 설명합니다. 이 방법은 연속 Percoll 기울기와 백혈구 - 농축 레이어를 선택하고 정화 불연속 Ficoll 기울기를 사용합니다. 절연 leukocytes 그때 흐름 cytometric 측정에 의해 특징 수 있습니다.

비디오: Isolation of Brain-infiltrating Leukocytes

Isolation of Functional Cardiac Immune Cells

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 include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

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 ...

연구

면역학 및 감염병학

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

에서 뇌 - 분리 된 백혈구의 분리 및 분석<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 마우스

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

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 ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

인간 백혈구 및 혈소판의 분리 및 평가를위한 프로토콜 : 생물 에너지와 산화 버스트

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

인간의 산모 - 태아 인터페이스에서 백혈구의 분리

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

산모 - 태아 인터페이스에서 쥐의 조직에서 백혈구의 분리

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating - 폐 백혈구의 선택적 수확

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-간 백혈구의 선택적 수확

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...