저자는 기술 지원을 위해 양을 리아나 Mackiewicz 감사드립니다. 이 작품은 빅토리아 주 정부 운영 인프라 지원 및 호주 정부 국립 보건 의학 연구위원회 IRIISS 및 프로젝트 기금 1,031,212을 통해 가능하게되었다.

1. P.있는 마우스의 감염 berghei - ANKA <ol><li> 녹이 cryopreserved P.의 나누어지는 berghei ANKA pRBC. </li><li> 두 손으로 구속 기법을 사용하여 뇌성 말라리아에 저항력이 BALB / C 공여 마우스를 (8-12주짜리) 구속. 1 ML의 인슐린 주사기 (28G 바늘을)를 사용 pRBC의 100-200 μl와 마우스를 주입. 일상적으로, 1-2 기증자 마우스가 주입되어 있습니다. </li><li> 후 감염 4~5일에서 (PI) 케이지에서 기증자 마우스를 제거하고 워크 스테이션이나 일회용 작업 패드에 놓으십시오. </li><li> 부드럽게 꼬리의 끝 부분에 마우스를 구속하고 작은 가위를 사용하여 (약 1mm) 꼬리 끝을 잘라. 또는 25 G 바늘을 사용하여 작은 꼬리 구멍이 수행 될 수 있습니다. </li><li> 불투명 엔드 현미경 슬라이드의 끝 부분 마우스의 꼬리 정맥에서 혈액 한 방울을 넣으십시오. </li><li> 45 ° 각도에 두 번째 슬라이드 (확장기)를 놓고 피 드롭으로 다시. 피가 가장자리를 따라 펼쳐지되면, 확장기 - 슬라이드 이브를 밀어핏자국을 할 수있는 현미경 슬라이드에 걸쳐 nly. 비방은 건조한 공기 할 수 있습니다. </li><li> 10 분에 신선하게 조리 된 Giemsa에서 슬라이드를 착색 한 후 30 초 100 %의 메탄올에 피 묻은 얼룩을 수정합니다. </li><li> 1 분에 물을 실행에 핏자국을 씻어, 공기 건조 할 수 있으며 현미경 (100x, 석유 침적)에서 슬라이드를 검사합니다. 1000 적혈구 내에서 pRBC을 열거하고 %의 parasitemia를 계산합니다. 그들이 실험 동물의 감염 출혈이되기 전에 기증자 마우스는 2.5-5 % 사이 parasitemia 수준에 도달해야합니다. </li><li> heparinized 마이크로 혈소판 모세관을 사용하여 복고풍 궤도 출혈에 의해 기증 마우스에서 혈액을 수집합니다. 모든 실험은 월터 엘리자 홀 연구소 및 동물 윤리위원회의 요구 사항을 준수 수행됩니다. 지역 동물 윤리위원회의 요구에 따라 다른 출혈이 절차를 사용할 수 있습니다. 1X10 6 pRBC/0.2 ML의 최종 농도에 RPMI 매체에서 혈액 필요한 양을 분해. 그을 고려일반 마우스 혈소판 ~ 6x10 9 적혈구 / 1 ML입니다. </li><li> intraperitoneally (IP) 1x10 6 pRBC/0.2 ML을 주입하여 실험 C57BL / 6 마우스 (8-12주 이전)를 감염. </li><li> 감염된 쥐 parasitemia 수준을 모니터링하려면 단계 1.3-1.8를 따르십시오. Parasitemia는 하루에 2 ~ 3 파이부터 2-3일 모든 결정해야 </li><li> 쥐가 hunched 모양과 같은 매니페스트 수도 C57BL에서 / 6 심한 말라리아의 증상은, 모피 및 낮은 활동을 주름 옷깃이 달린. 일부 마우스는이 단계에서 복구 할 수 있지만, 자기 righting 반사 손실에 진행은 돌이킬 수없는 질병을 나타냅니다과 동물은 euthanized해야합니다. </li></ol> 2. 마우스 및 뇌 추출의 Intracardial 재관류 <ol><li> 60cm 열 스탠드 상단에 부착 된 열 홀더에 500 ML 저장소를 확보하여 수동 중력 재관류 시스템을 조립. 저수지의 바닥 끝 부분에 플라스틱 튜브를 연결하고 버퍼 흐름을 제어 할 수있는 튜브 클램프를 고정하십시오. 23 G 바늘에 튜브를 연결합니다. FilPBS (22 ° C에 보관)과 저수지까지 내 말은, 클램프 및 모든 기포를 제거하는 튜브를 통해 실행하는 버퍼를 허용 엽니 다. </li><li> 안락사시켜야 P. CO 2 흡입하여 berghei ANKA에 감염된 마우스. 페달, 궤도 및 호흡기 반응의 부재에 의해 죽음을 확인합니다. </li><li> dorsally 플라스틱 트레이에 포함 된 스티로폼 분해 보드에 뒷다리와 앞 손으로 euthanized 마우스를 핀. 지역 동물 윤리위원회 요구 사항 마취에 따라 intracardial pefusion의 사전 개시를 관리 할 수​​있다. </li><li> 70 %의 에탄올과 복부 측면을 닦아주십시오. </li><li> 흉강을 노출하는 중간 선을 따라 피부를 열고 대형 가위와 집게를 사용하십시오. 측면에 접어 핀 피부. </li><li> 고급 집게로 가슴을 잡고 가로막을 절감하고 가슴의 양쪽을 따라 갈비뼈를 severing. 대용량의 혈관을 손상 않도록주의보십시오. </li><li> 머리를 느슨하게 옆에있는 흉골에 의해 흉곽을 고정. </li><li> 심실의 지혜를 개최H 고급의 포셉, 고급 가위로 조심스럽게 절개 우심방. </li><li> PBS가 실행되는 동안 오름차순 대동맥으로 왼쪽 심실에 비중 재관류 시스템에 부착 23G 바늘을 삽입합니다. 바늘 끝의 0.5 cm를 삽입합니다. </li><li> 5 분 또는 유출 될 때까지 마우스를 Perfuse 것은 분명하다. </li><li> 고정 해제 마우스와 복부에 놓여 있었다. 70 %의 에탄올과 머리를 닦아주십시오. </li><li> 큰 가위를 사용하여, 그냥 자궁 경부 척추 영역 위에 몫을합니다. </li><li> 중간 꼬리 - 뱃 부리 장식이있는 컷이 두개골을 노출 떨어져 두개골의 기지에서 시작, 피부 껍질을 벗기면하기 위해 고급 가위를 사용합니다. </li><li> 머리 개최, 각 궤도 구멍에 작은 가위 블레이드를 배치하고 궤도 사이의 몫을합니다. </li><li> 시상 봉합을 따라 길이 방향 절단을 확인하고 조심스럽게 바깥쪽으로 각각의 뇌 반구에있는 두개골 껍질을 벗기면. </li><li> 주걱 사용하여 부드럽게 뇌를 들고 RPMI 매체를 포함하는 10 ML의 원심 분리기 튜브에 넣습니다. </li></ol>3. 뇌 - 분리 된 백혈구의 절연 (BSL) <ol><li> 안전 캐비닛에서 근무 곳 갓 RPMI 조직 문화 매체 3-5 ML을 포함하는 배양 접시에있는 스테인리스 스틸 셀 스트레이너 (40-60 메쉬 크기)의 상​​단에 뇌를 수확. </li><li> 작은 조각으로 뇌 조직을 잘라 버릴거야. </li><li> 크리스탈 플런저를 사용하여 셀 스트레이너를 통해 뇌 조직의 작은 조각을 밀어 넣습니다. </li><li> 4에 10 분 ° C.에 250 XG에서 10 ML 튜브와 원심 분리기에 뇌 homogenate를 전송 </li><li> 0.05 % Collagenase D 및 2U/ml DNAse I.를 포함 RPMI 매체의 3 ML에서 펠렛을 분해 </li><li> 실온에서 30 분을위한 혼합물을 회전합니다. 70 μm 나일론 셀 스트레이너 및 /를 통해 혼합물을 눌러 5 분 동안 얼음에 잠복기에 의한 세포 파편을 제거합니다. </li><li> 실온에서 20 분 400 XG (브레이크)에서 7 ML 30 % Percoll 쿠션과 원심 분리기에 뇌 homogenate를 놓는다. </li><li> 5 분에 대한 얼음에 붉은 혈액 세포 용해 버퍼와 품다 1 ML에 Resuspend 펠릿intracardial 재관류에 의해 뇌에서 제거 할 수 없습니다 자기편 pRBC을 lyse. </li><li> RPMI 매체 9 ML을 추가, 4 ° C.에 10 분을 위해 250 XG에서 세포와 원심 분리기를 씻어 </li><li> RPMI 매체와 Eppendorf 튜브에 전송 세포의 50-100 μl에 Resuspend BSL 함유 펠렛. </li><li> 가능한 세포의 식별 Trypan 파란색으로 셀 샘플의 5-10 μl 나누어지는 1시 2분을 희석. </li><li> hemocytometer를 사용하여 현미경으로 가능한 셀을 계산합니다. 6 일 파이 (Π) 이후, P.에서 berghei ANKA에 감염된 마우스는 20,000-100,000 BSLs을 복구 할 수 있습니다, 신경 증상을 표시합니다. </li></ol> 4. 여러 가지 빛깔의의 유동 세포 계측법에 의해 BSL의 Immunophenotyping <ol><li> 4에 10 분 ° C에 250 XG에서 세포를 원심 분리기 (PBS, 1 % 태아 송아지 혈청 (FCS), 2mm EDTA (에틸렌 다이아 민 테트라 초산)) 착색 버퍼 50 μl의 표면에 뜨는 및 resuspend 펠렛을 대기음. </li><li> anti-CD16/32 항체 (Fcblock)를 정화의 1 μl를 추가합니다. </li><li> 1 세포를 배양얼음에 0 분. </li><li> 얼룩 버퍼 1 ML과 세포를 씻으십시오. 4 ° C에서 10 분 동안 250 XG에서 세포를 원심 분리기와 표면에 뜨는을 대기음. </li><li> 필요한 형광 항체의 미리 정해진 최적의 dilutions 즉, 안티 - CD4, 항 CD8, 안티 TCR 및 안티 NK1.1를 포함하는 버퍼를 착색의 50 μl에 Resuspend 세포. </li><li> 얼음에 1 시간에 품다. </li><li> 얼룩 버퍼 1 ML과 세포를 씻으십시오. 4 ° C에서 10 분 동안 250 XG에서 세포를 원심 분리기와 표면에 뜨는을 대기음. </li><li> PBS 100 μl에 세포를 Resuspend. </li><li> 유동 세포 계측법 플라스틱 튜브에 세포를 전송합니다. </li><li> 흐름 cytometer를 사용, 적어도 5,000-10,000 이벤트를 취득. 필요에 따라 적절한 흠없는 셀 컨트롤과 fluorochrome 보상 샘플이 포함되어야합니다. </li><li> 적절한 유동 세포 계측법 소프트웨어를 사용하여 결과를 분석합니다. </li></ol>

<ol>
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관심 없음 충돌이 선언 없습니다.

BSL의 절연 및 분석은 특성화 및 실험 마우스 모델에서 조직 부상이나 감염에 대한 응답으로 뇌에 이주하는 염증 세포의 정량화 할 수있는 방법입니다. 장기 추출 및 후속 전지 분리하기 전에 혈액 구획의 제거를위한 intracardial 재관류 단계의 도입은 비 염증성 순환 백혈구와 염증 세포의 오염을 방지하기 위해 유용합니다. 이 염증 세포가 뇌 실질 5 침투하는 등 손쥐 뇌척수염 바이러스 등?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td></td><td></td><td></td><td> 솔루션 및 버퍼 </td></tr><tr><td> Giemsa의 다 쥐르 eosin의 메틸렌 블루 용액 </td><td> 머크 Millipore </td><td> 1.09204.0500 </td><td> 증류수에 희석 1시 10분 </td></tr><tr><td> RPMI 배지 </td><td></td><td></td><td> 마우스 tonicity </td></tr><tr><td> 마우스 tonicity PBS </td><td></td><td></td><td> 20 밀리미터 인산 나트륨, 0.149 NaCl, pH를 7.3 </td></tr><tr><td> 0.4 % Trypan 파랑 </td><td> 시그마 알드리치 </td><td> T-8154 </td><td> 1시 2분 희석 </td></tr><tr><td> Collagenase D </td><td> 생물 화학 워딩 턴 </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAse) I </td><td> 시그마 알드리치 </td><td> D4263-5VL </td><td> 소 췌장에서 </td></tr><tr><td> Percoll </td><td> GE 헬스 케어 </td><td> 17-0891-01 </td><td> PBS의 30 % 솔루션 </td></tr><tr><td> Ultrapure 트리스 </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> 염화 암모늄 (NH 4 망할 CIA) </td><td> AnalaR </td><td> 10,017 </td><td></td></tr><tr><td> 적혈구 용해 버퍼 </td><td></td><td></td><td> 17 MM 트리스, 14nM NH 4 CIA 같으니, pH를 7.2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) 나트륨 소금 </td><td> 머크 </td><td> 10093.5V </td><td> 0.1M, pH를 7.2 </td></tr><tr><td></td><td></td><td></td><td> 항체 conjugates </td></tr><tr><td> 안티 마우스 CD16/CD32 (FC 블록), 클론 2.4G2 </td><td> BD Pharmingen </td> &lt;TD&gt; 553142 </td><td> 50 μl 착색 버퍼 1 μl (0.5mg/50 ML) </td></tr><tr><td> FITC - 안티 - 마우스 CD4, 클론 H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-안티 - 마우스 NK1.1, 클론 PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5 - 안티 - 마우스 CD8, 클론 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC - 반 마우스 TCR-β, 클론 H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-안티 - 마우스 CXCR3, 클론 220,803 </td><td> R &amp; D 시스템 </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinylated - 안티 - 마우스 CCR5, 클론 C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Steptavidin - PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 및 재료 </td></tr><tr><td> SuperFrost 현미경 슬라이드 </td><td> Lomb Menzel - Gläser </td><td></td><td></td></tr><tr><td> 분해 집게, 가위 </td><td> 레다 Instrumente </td><td></td><td></td></tr><tr><td> 500 ML PBS 저수지 </td><td> Nalgene </td><td></td><td></td></tr><tr><td> 고무 튜빙 </td><td></td><td></td><td></td></tr><tr><td> 23G 바늘 </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> 세포 분리 키트는 금속 체를 포함하는 </td><td> 시그마 알드리치 </td><td> CD-1 </td><td></td></tr><tr><td> 70 μm 나일론 셀 스트레이너 </td><td> BD 팔콘 </td><td> 352350 </td><td></td></tr><tr><td> Hemocytometer </td><td> GmbH의 Neubauer </td><td> 717810 </td><td></td></tr><tr><td> 유동 세포 계측법 관 </td><td> BD 매 </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

우리는 P.의 뇌 혈관의 점착성의 염증 세포의 분리 및 특성에 대한 방법을 설명 berghei ANKA에 감염된 쥐. 실험 뇌성 말라리아의 유도, neurologic 증후군이 기생충 변형 결과를 따르게 마우스 변종의 감염 인간 1,2에서 Plasmodium falciparum로 인한 심각한 말라리아 recapitulates 특정 중요한 측면이. 혈액 단계 말라리아의 중년 여인 형태들이 혈관 내피 세포에 바인딩 할 수 있습니다 감염된 적혈구의 표면에 기생 단백질을 표현한다. 이 과정은 hypoxia와 haemorrhages 3 결과, 혈액의 흐름에 장애물을 유도하며 기생충 격리의 사이트에 염증 백혈구의 모집을 자극. 다른 감염, 즉 neutrotopic 바이러스 4-6 말라리아-parasitized 적혈구 (pRBC)뿐만 아니라 관련 inflamm 모두는 달리atory 백혈구는 혈관 내에서 오히려 뇌 실질에 침투보다 분리 된 상태로 유지됩니다. 따라서 비 염증 순환 세포, 장기의 추출 및 조직 처리하기 전에 감염된 - 쥐의 광범위한 intracardial 재관류로 분리 된 백혈구의 오염을 방지하는 것은 혈액 구획을 제거하는이 절차가 필요합니다. 재관류 후, 머리가 수확되고 작은 조각으로 해부 했어요. 조직 구조는 더욱 그 결과 뇌 homogenate 그때 myelin 및 기타 조직 쓰레기들로부터 뇌를 분리 백혈구의 분리 (BSL)을 할 수있는 Percoll 기울기에 centrifuged되어 Collagenase D 및 DNAse I.있는 효소 처리에 의해 방해된다. 절연 셀이 후 씻어 있으며, 유동 세포 계측법에 의한 후속 분석을 위해 형광 항체와 hemocytometer과 스테인드을 사용하여 계산. 이 절차는 염증성 백혈구의 종합적인 phenotypic 특성을 다시에있는 뇌로 마이그레이션 할 수 있습니다뇌졸중뿐만 아니라 바이러스 또는 기생충 감염 등 다양한 자극에 sponse. 방법은 또한 사전 임상 동물 모델에서 소설 항 염증성 치료의 평가에 유용한 도구를 제공합니다.

그림의 결과입니다. 이 쇼 비율과 perfused 또는 unperfused 말라리아에 감염된 그리고 나이브 제어 마우스의 뇌에서 발견 한 다른 BSL 인구의 절대 숫자. 프로토콜 텍스트에 표시된대로 절연 BSL는 PE - 반 NK1.1과 APC-반 TCR-β 항체 물들했다. 이전 결과 7-9에 의거하여 αβTCR + T 세포는 perfused 말라리아에 감염된 쥐 (6 일 PI)의 뇌에있는 BSL 수영장의 높은 비율을 구성. 이 인구는 크게 (그림 2A-B)...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

의 뇌 혈관의 점착성의 염증성 백혈구 분리하기위한 방법<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 쥐들이 설명되어 있습니다. 방법은 유동 세포 계측법에 의해 형광 항체와 후속 분석과 얼룩 후 정량화뿐만 아니라 절연 백혈구의 phenotypic 특성 수 있습니다.

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-analysis-brain-sequestered-leukocytes-from-plasmodium

Research

JoVE Journal

Immunology and Infection

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

12.7K 조회수.  The Walter and Eliza Hall Institute of Medical Research. 의 뇌 혈관의 점착성의 염증성 백혈구 분리하기위한 방법 Plasmodium berghei ANKA에 감염된 쥐들이 설명되어 있습니다. 방법은 유동 세포 계측법에 의해 형광 항체와 후속 분석과 얼룩 후 정량화뿐만 아니라 절연 백혈구의 phenotypic 특성 수 있습니다.

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

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 ...

연구

면역학 및 감염병학

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

약독 화와 피내 접종에 따라 마우스의 피부와 배수 림프절에서 골수 세포 분리<em&gt; 변형체</em&gt; 포자 소체

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...