우리는 초기 프로토콜을 속에에 레 사노의 공헌을 인정합니다. Ad&#39;A. 유전 및 유전자 치료에 의자를 부자 어린이 (JFC)의 보석상을 보유하고 있습니다. 이 작품은 NIH 보조금 GM60905, DK52025 및 CA021764, 그리고 미국의 레바논 시리아 관련 자선 단체 (ALSAC)에 의해 부분적으로 재정 지원을했습니다.

<html> 다음 프로토콜은 마우스 뇌에서 MAMs과 보석의 절연 및 정화를위한 것입니다 솔루션은 미토콘드리아, MAMs와 보석의 절연이 필요 분류 원유 mitochondrial 준비를 얻기 위해 : 솔루션 A : 0.32 M의 자당, 1 MM NaHCO 3, 1 MM MgCl 2, 0.5 MM CaCl 2 + 프로테아제 저해제 (필요에 따라, 신선한 추가) 솔루션 B : 0.32 M의 자당, 1 MM NaHCO 3 + 프로테아제 저해제 (필요에 따라, 신선한 추가) MAMs 절연 : 절연 매체 : 250 MM 마니 톨, 5 밀리미터 HEPES의 산도 7.4, 0.5 MM EGTA, 0.1 % BSA 그라디언트 버퍼 : 225 MM 마니 톨, 25 밀리미터 HEPES의 산도 7.5, 1 ㎜ EGTA, 0.1 % BSA (최종 농도) 보석 절연 : 내용 &quot;&gt; GEM 추출 버퍼 : 25 MM HEPES 산도 7.5, 0.15 M NaCl, 1 % 트리톤 X-100 + 프로테아제 저해제 (필요한 경우, 신선한 추가) GEM Solubilising 버퍼 : 50 MM 트리스 - HCL, pH를 8.8, 5 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 % SDS + 프로테아제 저해제 (필요에 따라, 신선한 추가). 1. 첫 번째 단계 Subcellular 분류 : 핵의 제거, Unlysed 세포와 세포 파편 <ol><li> CO 2 챔버에 마우스를 안락사시켜야. </li><li> 즉시 뇌를 제거 절반, 얼음에서 2 ML 튜브에 넣고 무게. </li></ol> 참고 : 전체 절차를 통해 뇌 절반은 별도의 유지. 다음은 하나의 뇌의 절반의 치료에 적용됩니다. <ol start="3"><li> 한 ML 차가운 솔루션 A.이 큰 통관 유봉의 15 총 스트로크와 균질화가 포함 된 사전 차가운 두 ML 유리 다운스 호 모지 조직 분쇄기의 장소 반 뇌. </li><li> 얼음에 15 ML 팔콘 튜브에 전송하고 희석10 권 w / V, 예를 들어 0.225 g에 2.25 ML까지 homogenates. </li><li> 4에 10 분에 1,400 XG에서 샘플을 원심 분리기 ° C. </li><li> 조심스럽게 얼음에 표면에 뜨는과 30 ML 둥근 바닥 유리 원심 분리기 튜브로 전송을 제거합니다. 펠렛을 방해하지 않도록하십시오. </li><li> 솔루션 같은 10 권의 펠렛을 Resuspend. 작은 통관 유봉, 한 번에 1 ML의 3-6 뇌졸중과 같은 그라인더에 균질화. </li><li> 4에 10 분 ° C.에 710 XG에 신선한 15 ML 팔콘 튜브와 원심 분리기로 전송 핵 및 세포 파편의 펠렛에있는이 결과. </li><li> 주의 단계 1.6에서 저장 한 표면에 뜨는있는 표면에 뜨는과 수영장을 제거합니다. </li></ol> 2. 두 번째 단계 : 조잡 미토콘드리아 절연 <ol><li> 4에 10 분에 13,800 XG에서 supernatants을 원심 분리기 ° C. </li><li> 얼음에 울트라 클리어 Beckman의 원심 분리기 튜브를 표면에 뜨는 전송 </li><li> Resuspend 펠릿 10 권 솔루션에. 작은 통관 유봉, 한 번에 1 ML의 3-6 뇌졸중과 같은 그라인더에 균질화. </li><li> 단계 2.1로 원심 분리기. </li><li> 단계 2.2-2.4를 반복합니다. </li><li> 그 결과 펠렛은 풍부한 mitochondrial 분수입니다. 풀 supernatants (세포 기질 및 ER), parafilm으로 커버하고 얼음 계속. </li><li> 작은 통관 유봉 6 획으로 펠렛을 Resuspend, 4.8에 ML / g는 신선한 homogeniser에 솔루션 B의 (g는 원래 뇌의 무게라고합니다). </li><li> 유리 파스퇴르 피펫 사용하여, 신중하게 다음과 같이 관의 맨 ​​아래에이어서 추가 더 거품이 형성되지 않습니다 보장 울트라 클리어 Beckman의 원심 분리기 튜브에서 불연속 자당의 그라디언트를 준비 : Resupended 펠렛 (0.32 M의 자당) 3 ML 850 밀리미터 자당 (1 MM NaHCO 3) 3 ML 1 M의 자당 (1 MM NaHCO 3) 3 ML 1.2 M의 자당 (1 MM NaHCO에서3) </li><li> 4 ° C.에서 2 시간에 82,500 XG에 원심 분리기 그라디언트의 끝에서 그 결과 분리는 세 밴드와 펠렛 생산합니다 : - 2) ER, Golgi, 플라즈마 멤브레인 (0.85 M - 1 M 인터페이스) 0.85 M 인터페이스) 1) myelin 및 기타 막 오염 물질을 (0.32 M, 3) synaptosomes (1 M - 1.2 M 인터페이스), 4) 미토콘드리아 원유 (펠렛) 이후 정화 단계에 사용되는 </li></ol> 3. 미토콘드리아 - 관련 ER 막의 절연, MAMs <ol><li> 갓 추가 단백 분해 효소 억제제를 포함하는 두 ML 절연 매체에 하나의 뇌 절반에서 미토콘드리아 갓 고립 된 원유를 Resuspend. </li><li> 그라데이션 버퍼 8 이분의 일 뇌 당 ML, 그리고 울트라 클리어 Beckman의 원심 분리기 튜브의 장소와 30 % Percoll 기울기를 준비합니다. </li></ol> * 참고 : D 후 정확한 최종 농도를 달성하기 위해 (1.43 배) 기울기 버퍼가 더 집중하기30 % Percoll을 iluting. <ol start="3"><li> 준비된 기울기 상단에 레이어 mitochondrial 정지는 천천히 모든 거품을 방지합니다. </li><li> 30 분, 4 ° C.에 95,000 XG에 원심 분리기 </li><li><ol class="lroman"><li> 유리 파스퇴르 피펫으로 FIRST 중공업 비율 (낮은 밴드)를 제거하고 신선한 왕복 아래 유리관에 전송, 얼음. </li><li> 또 다른 유리 파스퇴르 피펫 두 번째는 빛 분수 (위 밴드)를 제거하고 얼음에 별도의 신선한 왕복 아래 유리관에 전송할 수 있습니다. </li></ol></li><li> 4에 10 분에 6,300 XG에서 10 ML 절연 매체와 원심 분리기와 단계 3.5 수집 된 두 분수 ° C.를 희석 </li><li> 단계 3.6에서 얻어진 중공업 비율의 표면에 뜨는 폐기하십시오 다른 10 ML 절연 매체로 펠렛을 resuspend과 단계 3.6로, 다시 원심 분리기. 그 결과 펠렛은 순수 mitochondrial 분율 될 것입니다. </li><li> 빛 Frac에서 표면에 뜨는을 전송기가 얼음에 울트라 클리어 Beckman의 원심 분리기 튜브에 단계 3.6에서 얻은. 펠렛을 폐기하십시오. </li><li> 1 시간, 4 ° C.에 100,000 XG의 튜브와 원심 분리기를 작성 단계 3.8에서 얻어진 표면에 뜨는에 충분한 절연 매체를 추가 그 결과 펠렛은 MAMs 분수됩니다. 조심스럽게 표면에 뜨는을 제거하고 폐기합니다. </li></ol> 참고 :이 단계에서, 여러분은 또한 1 시간, 4 십만 XG에 원심 분리기 수 있습니다 ° C 단계 2.6에서 저장 한 표면에 뜨는. 펠렛은 ER 분수이어야하며 Cytosolic 일부를 표면에 뜨는 것입니다. 4. Glycosphingolipid 강화 Microdomains, 보석 추출 <ol><li> 얼음에 20 분에 추출 버퍼 500 μl-1 ML에 순수 미토콘드리아 및 / 또는 이맘 분수를 Lyse. </li><li> 4에서 2 분에 15,300 XG에 lysates을 원심 분리기 ° C. </li><li> supernatants (트리톤 X-100 수용성 물질)와 다시 원심 분리기 알약을 수집2 분에 용해 물질을 남아 제거합니다. (트리톤 X-100은 미토콘드리아 추출 및 / 또는 트라이 튼 X-100 추출 MAMs). </li><li> 버퍼 Solubilising에서 알약을 Solubilize. 이 solubilized 자료는 GEM의 분수를 상징하며 추가적인 분석을 위해 사용할 수 있습니다. </li></ol>

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저자는 모두 신고 할 관심의 충돌이 없습니다.

세포 멤브레인 간 또는 세포 소기관과 세포의 플라즈마 막 사이의 접촉의 사이트는 기본 세포 프로세스에 동적 신호 플랫폼을 나타냅니다. 생리학 및 병리학 조건 모두에 따라 자신의 기능과 구성의 정확한 특성은 안정적이고 재현 정화 프로토콜이 필요합니다. 여기에 설명 된 방법은 특별히 성인 마우스의 뇌에서 MAMs와 해당 보석의 절연 및 정화를 위해 우리 실험실에서 최적화되었습니다. 이 프로토콜이 성공적으로 어린이 4 neurodegenerative 신진 대사 질환에 neuronal 세포 사망에 이르는 + 불균형을이 microdomains을 지정하고, 칼슘 2의 apoptotic 경로의 하류를 기초 분자 effectors의 식별을 위해 구현되었습니다.

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td></td><td></td><td></td><td> 시약 </td></tr><tr><td></td><td></td><td></td><td> 분류 </td></tr><tr><td> 자당 </td><td> 피셔 과학 </td><td> S5-500 </td><td></td></tr><tr><td> 나트륨 중탄산염 </td><td> 시그마 - 알드리치 </td><td> S-5761 </td><td></td></tr><tr><td> 염화 마그네슘, Hexahydrate </td><td> 피셔 과학 </td><td> BP214-500 </td><td></td></tr><tr><td> 칼슘 염화물, 이수화 </td><td> 시그마 - 알드리치 </td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> 이맘 </td></tr><tr><td> D-마니 톨 </td><td> 시그마 - 알드리치 </td><td> M9546-250G </td><td></td></tr><tr><td> Hepes </td><td> 피셔 과학 </td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td> 시그마 - 알드리치 </td><td> E4378-250G </td><td></td></tr><tr><td> BSA, 분수 V, 열 충격은 Lyophilizate </td><td> 로슈 </td><td> 03-116-964-001 </td><td></td></tr><tr><td> Percoll </td><td> GE </td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> GEM </td></tr><tr><td> 트리톤 X-100 </td><td> 시그마 - 알드리치 </td><td> T9284-500 ML </td><td></td></tr><tr><td> 나트륨 염화물 </td><td> 피셔 과학 </td><td> S271-3 </td><td></td></tr><tr><td> 트리스베이스 </td><td> 로슈 </td><td> 03-118-142-001 </td><td></td></tr><tr><td> HCL </td><td> 피셔 과학 </td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 피셔 과학 </td><td> BP120-500 </td><td></td></tr><tr><td> 나트륨 Dodecyl의 황산 (SDS) </td><td> 피셔 과학 </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> 공통의 </td></tr><tr><td> 전체 단백 분해 효소 저해제의 태블릿, EDTA (에틸렌 다이아 민 테트라 초산) - 무료 </td><td> 로슈 </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> 2 ML 조용한 전체가 유리로 조직 분쇄기 </td><td> 킴블 체이스 </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ML의 폴리 프로필렌 원뿔형 원심 분리기 튜브, BD 매 </td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30 ML 라운드 - 하단 유리 원심 분리기 튜브 </td><td> 킴블 체이스 </td><td> 45500-30 </td><td></td></tr><tr><td> 15 ML 라운드 - 하단 유리 Centrifuge 튜브 </td><td> 킴블 체이스 </td><td> 45500-15 </td><td></td></tr><tr><td> Ultracentrifuge 튜브, 울트라 클리어, Thinwall, 14x89 mm </td><td> Beckman - 보습 바로 앞에 달린 풀 베는 날 </td><td> 344059 </td><td></td></tr><tr><td> Parafilm </td><td> 콜 - Parmer </td><td> PM996 </td><td></td></tr><tr><td> 일회용 붕규산 유리 파스퇴르 (Pasteur) Pipets, 9 &quot; </td><td> 피셔 과학 </td><td> 13-678 - 20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

세포 세포 소기관은 모양과 셀 별 고유 및 외부 신호를 받게 아르 구성을 변화와 고도의 동적 구조입니다. 그들의 멤브레인은 종종 신호 분자와 막 구성 요소 1,2,3,4의 교환을위한 허브가 정의 연락처 사이트에서 juxtaposed 있습니다. endoplasmic 소포체 (ER)와 + 채널이 미토콘드리아 관련-ER 멤브레인 또는 MAMs 4,5,6로 알려져 있습니다 IP3에 민감한 칼슘 2 개구에서 미토콘드리아 사이에 형성되는 간 organellar 막 microdomains. 단백질 / 지질 조성과 이러한 멤브레인 연락처 사이트의 생화학 특성을 광범위하게 특히 세포 내 칼슘 2 + 4,5,6을 조절에서의 역할과 관련하여 연구되었습니다. ER은 +,이 용량에서 칼슘이 세포의 하류 공정의 무수한을 조절 세포 칼슘 (2)의 기본 저장소 역할을 + 신호, incl포스트 병진 단백질 접힘, 단백질 maturation7을 uding. 미토콘드리아, 다른 한편으로,이를 통해 하류 칼슘 2 + 불균형 4,8의 apoptotic 경로의 개시를 방지 cytosolic 칼슘 2 + 농도를 버퍼링하여 칼슘 2 + 항상성을 유지하고 있습니다. MAMs의 동적 특성은 그들에게 이상적인 사이트는 칼슘 2 + 신호와 mitochondrial 칼슘 2 + 농도, 지질 생합성 및 수송, 에너지 신진 대사와 세포 생존 4,9,10,11,12 규제 등의 기본적인 세포 메커니즘을 해부 할 수 있습니다. 여러 프로토콜은 간 조직 및 배양 세포 13,14에서 이러한 microdomains의 정화에 대한 설명되었습니다. 계정에 이전에 다음 ID로 출판 방법을 복용, 우리는 성인 마우스의 뇌에서 미토콘드리아와 MAMs의 절연을위한 프로토콜을 적응하고 있습니다. 이 절차에 우리는 추가 정화 단계, 즉 트리톤의 X100 추출을 추가하는 전자nables MAMs의 glycosphingolipid 강화 microdomain (GEM) 분수의 고립. 이러한 GEM의 준비가 플라즈마 막 나 다른 세포 세포막에서 유래 caveolae과 지질 보트와 여러 단백질 구성 요소를 공유하고 수용체 단백질의 클러스터링에 대한 포인트를 수집처럼 단백질 단백질 상호 작용 4,15에 대한 작동하도록 제안하고 있습니다.

우리가 안전하게 MAMs, 보석 및 마우스의 뇌에서 mitochondrial 분수의 절연 및 정화를 위해 추천 할 수 있습니다이 프로토콜을 사용하여와의 경험을 바탕으로. 명시된 절차는 매우 재현과 일치합니다. 그림 1에서 우리는 Percoll 기울기 (단계 3.4)의 방법으로 순수 미토콘드리아와 MAMs 층의 대표 이미지를 보여줍니다. 이맘 분율은 미토콘드리아 위의 확산과 폭 넓은 대역을 구성하는 동안 포함 된 정의, 우유 밴드, ultracentrifuge 튜브의 하단에있는 미토콘드리아 (미토 P) segregates를 정화. 개별 기울기 밴드의주의 회복 후, 정화 분수는 용해 버퍼에 resuspended 아르 SDS의 polyacrylamide 젤에서 분리 PVDF 막을에 blotted. Blots은 다음 분수와 단백질 성분의 순도를 확인합니다 특정 단백질 마커에 대한 항체의 배터리로 탐색합니다. 그림 2A는 cytos의 분포를 보여줍니다고립 된 MAMs과 mitochondrial 분수에 olic ER과 mitochondrial 표시. 순수 미토콘드리아 준비는 ER과 cytosolic 마커 모두의 결여해야합니다. 이맘 연락처 사이트에서 ER과 mitochondrial 멤브레인 사이의 긴밀한 동격이 정화 준비에​​ calreticulin (ER 마커)와 탐 - 20 (미토콘드리아 마커)의 존재를 설명합니다. 이 MAMs에게 특정 마커 FACL4와 PACS2,뿐만 아니라 IP3R-1과 GRP75 4와 같은 이러한 도메인 내에서 풍부한 다른 마커를 사용하는 것도 가능합니다. MAMs은 또한 보석을 얻기 위해 트라이 튼 X-100으로 추출 할 수 있습니다. 그림 2B와 같이 지질 보트 및 / 또는 caveolae의 구성 요소가 포함이 microdomains는 caveolin-1 긍정적입니다. <img src="/files/ftp_upload/50215/50215fig1.jpg" alt="그림 1"/> 그림 1. mitochondrial - 관련 ER 막 (MAMs)의 레이어링의 사진을 표시하고 순수mitochondrial 분수 (미토-P). <img src="/files/ftp_upload/50215/50215fig2.jpg" alt="그림 2"/> 그림 2. A) 서양 blots은 순결과 cytosolic, ER 및 Cytosolic의 mitochondrial 마커 (cyto)의 분포를 확인하는 실행 표시 - ER-, 순수 mitochondrial 분수 (미토-P) -.의 엄만 - 분수 B) 서양 blots MAMs로부터 격리 glycosphingolipid 풍부한 microdomains (보석)와 트리톤 X-100 추출 (트라이 튼 extr. MAMs) 분수를 정화.

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

이 절차는 성인 마우스 뇌 미토콘드리아 - 관련 ER의 멤브레인 또는 MAMs 및 MAMs과 mitochondrial 준비에서 glycosphingolipid 강화 microdomain 분수에서 분리하는 방법을 보여줍니다.

미토콘드리아 - 관련 ER의 멤브레인 (MAMs)와 Glycosphingolipid 강화 Microdomains (보석) : 마우스 뇌에서 절연

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Nanyang Technological University

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Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

24.3K 조회수.  St Jude Children's Research Hospital. 이 절차는 성인 마우스 뇌 미토콘드리아 - 관련 ER의 멤브레인 또는 MAMs 및 MAMs과 mitochondrial 준비에서 glycosphingolipid 강화 microdomain 분수에서 분리하는 방법을 보여줍니다.

비디오: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

포스트 - 나탈 Murine 조직의 공동 문화를 Myelinating 풍부한 Oligodendrocyte 문화와 Oligodendrocyte / 신경 세포의 유도

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

배아 전뇌에서 내피 세포의 분리 및 문화

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

해마 조각에서 CA1 핵 농축 분획의 분리는 Synapto 핵 메신저 단백질의 활동에 의존하는 핵 가져 오기를 공부합니다

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

신선한 인간의 뇌 조직에서 뇌 모세 혈관의 절연

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

절연 및 애벌레 Zebrafish 두뇌에서 신경 세포, 대 식 세포 및 Microglia RNA 추출

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

깊은 단세포 RNA 염기서열 분석에 대한 하나의 성인 마우스 뇌 반구에서 영역 별 Microglia의 격리

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

산후 (P1−3) 마우스 신경성 틈새에서 신경구의 격리 그리고 확장

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

1 차 배아 마우스 중뇌 도파민 뉴런에서 사전 형성 된 피브릴 유도 α-시뉴클레인 축적 연구

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...