우리는 편집 지원 Jonna 엘리스 감사드립니다. 정신 건강의 국립 연구소에서 : 설명이 프로젝트는 보너스 번호 R01MH079751 (F. Peruzzi PI)에 의해 지원되었다. 내용은 전적으로 저자의 책임이며 반드시 정신 건강의 국립 연구소 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

1. 폴리-D-라이신 (PDL) : 준비 <ol><li> 1 MG / ML의 주식 솔루션을 얻기 위해 PDL의 5 MG로 멸균 ddH 2 O 5 ML을 추가합니다. </li><li> 여러 차례 pipetting하여 주식 솔루션을 섞는다. </li><li> 즉시 사용하거나 2-8로 폴리-D-라이신 솔루션을 저장할 ° C. </li></ol> 2. 폴리-D-라이신 (PDL) : 코팅 플라스틱 세포 배양 접시 <ol><li> 멸균 ddH 2 O을 갖춘 10 μg / ML의 최종 농도로 PDL 주식 솔루션을 희석. </li><li> 문화 표면적 (60 밀리미터 접시 3 ML)를 커버하는 60 밀리미터 접시에 피펫 충분 솔루션입니다. </li><li> 문화 표면에도 코팅을 보장하기 위해 부드럽게 신난다. </li><li> 하룻밤 실온 (RT)에 코팅 번호판을 품어. </li><li> 다음날, 보통 절개의 날, 흡인에 의해 폴리-D-라이신 솔루션을 제거하고 멸균 ddH 2 O를 3 ML로 간단히 씻어 이 단계를 반복합니다. 두 번째 세척 후 흡인에 의해 완전히 물을 제거합니다. </li><li>플레이트는 최대 3 주가에 대해 4 ° C에 저장할 수 있습니다. </li></ol> 3. 폴리-D-라이신 (PDL)과 Laminin : 유리의 준비 및 코팅 투 챔버 슬라이드 <ol><li> 각각 10, 5 μg / ML의 최종 농도로 멸균 ddH 2 O의 혼합 PDL (1 밀리그램 / ML)과 laminin (1 밀리그램 / ML) 증권 솔루션. </li><li> 유리의 우물 두 챔버 슬라이드 문화 표면적 (이 아니라 유리 두 챔버 슬라이드의 각도 1 ML)을 충당할로 피펫 충분한 솔루션입니다. </li><li> 문화 표면에도 코팅을 보장하기 위해 부드럽게 신난다. </li><li> 하룻밤 RT에서 코팅 번호판을 품어. </li><li> 다음 날에는 열망을 통해 폴리-D-라이신-Laminin 코팅 용액을 제거하고 멸균 ddH 2 O에 1 ML로 두 번 간단히 씻고 두 번째 세척 후 흡인에 의해 완전히 물을 제거합니다. </li><li> 상공 회의소 슬라이드는 최대 3 주가에 대해 4 ° C에 저장할 수 있습니다. </li></ol> 참고 : 모든 유리 챔버 슬라이드가 따라와에게 입혀져 수g이 프로토콜. 각 슬라이드 컨트롤을 테스트 실험적인 설정을 (예를 들어 치료 비교 치료, untransfected 대 transfected) 제공하기 때문에 우리는 종종 두 챔버 슬라이드를 사용합니다. 4. 의 연결을 해부와 문화 <ol><li> 37 ° C의 물을 욕조에 다음과 같은 시약을 따뜻하게 : <ul type="disc"><li> 원래 100 ML 병에 TrypLE 익스프레스. </li><li> Neurobasal/B27 완전한 매체 (표 I 참조). 예열 볼륨 (10 60mm 도금 요리 등 30 ML) 도금이 될 접시의 수에 따라 달라집니다. </li></ul></li><li> 네 60mm 배양 접시 15 ML BD 팔콘 높은 선명도의 폴리 프로필렌 원뿔 튜브 13 ML에서 차가운 최대 절전 E 솔루션 3 ML을 추가합니다. </li><li> 세 100mm 문화 요리 각 차가운 해부 매체 (표 II, 박사 Olimpia Meucci, 개인 통신 참조) 25-30 ML을 추가합니다. 매체의 큰 볼륨을 포함하는이 번호판은 즉시 그 후 배아를 씻어 사용됩니다amniotic 주머​​ 니나 (단계 4.7 및 4.8)에서 제거. </li><li> 실험실 동물의 동물 애호 관리 및 사용에 대한 보건 서비스 정책에 따라 그리고 institutionally 승인된 동물 보호 아래에서 CO 2가 E17 초과 임신 쥐를 안락사와 프로토콜을 사용합니다. </li><li> 70 % EtOH로 낮은 복부를 뿌려서 자궁과 태아를 노출 가위로 피부와 근육을 통해 medially 했네요. </li><li> 모든 fetuses를 제​​거하고 차가운 해부 매체 (25-30 ML 단계 4.3 참조) 초과를 포함하는 무균 100 mm 접시에 그들을 배치. </li><li> amniotic SAC에서 가위의 작은 쌍을 사용하여 배아를 잘라 차가운 해부 매체를 포함하는 두 번째 100 mm 접시에 그들을 배치. </li><li> 5-10초 대한 부드럽게 틸팅 100-mm 요리하여 실온에서 배아 씻으십시오. 그 다음 해부 매체를 포함하는 세 번째 100mm 접시에 씻어서 배아를 전송합니다. 과도한 중간의 두 세차장은 일반적으로 혈액의 모든 흔적을 제거하기에 충분합니다. 다만, NECessary, 차가운 해부 매체 25-30 ML을 포함하는 새로운 100 mm 접시를 사용하여 한 번 더 씻는다. </li><li> stereomicroscope과 곡선 포셉 사용하여 피부와 두개골을 철수하여 각 쥐의 배아의 두뇌를 추출합니다. 감기 최대 절전 엑스타 60 mm 요리 (일반적으로, 요리 당 더이상 5 달러 뇌를 포함) 얼음이 번호판을 보관 중 하나로 전체 두뇌를 놓습니다. </li><li> 한 번에 한 접시를 타고, 해부 현미경, 반구를 분리하고 midbrain과 meninges 제거 대뇌 cortices을 분리할. </li><li> 옵션 : 중간선을 따라 커팅 두뇌가 hippocampi를 추출하고, hippocampal 신경 세포를 분리하려면 아래 절차를 따르십시오. </li><li> 모든 해부가 완료될 때까지 얼음 대뇌 cortices를 남겨 추위 최대 절전 E. 13 ML을 포함하는 15 ML 맑은 원뿔 튜브의 모든 해부 cortices를 수집합니다. 그들의 작은 크기로 인해 해부 hippocampi 대신 15 ML 튜브의 1.5 ML Eppendorf 튜브에 수집 할 수 있습니다. 또는이 단계 cortices에서 원하는 경우hippocampi가 1 ML을 포함 cryotube의 유리병에 삽입하실 수 있습니다 최대 절전 E + 2% B27 + Gentamicin (50 μg / ml) 쇼핑은 2-4 cortices이나 유리병 당 2-4 hippocampi의 비례 + Fungizone (250 NG / ML). 뇌 조직은 한 개 주 (이후 시간은 아직 테스트되지 않은)에 대해 어두운 4 ° C에 저장할 수 있습니다. 필요한 경우, 최대 절전 E를 포함하는 15 ML 관으로 뇌 조직을 전송하고 뉴런을 분리하기 위해 아래의 프로토콜을 따르도록 미세 집게를 사용합니다. </li><li> 조직 문화 후드에 튜브를 전송합니다. cortices는 튜브의 바닥에 정착 후 신중하게 뜨는을 제거하도록 허용합니다. </li><li> , 15 ML 원뿔 튜브에 신선한 최대 절전 전자 13 ML 추가 cortices는 튜브의 아래쪽에 정착 허용하고 신중하게 뜨는을 제거합니다. 이 2 단계로 여러 번 반복하고 마지막 세척 후 신중하게 모든 미디어를 제거합니다. </li><li> 따뜻한 TR의; 효소 1-2 ML을 (hippocampi 절연을위한 더 적은을 사용 cortices의 숫자에 따라 다름) 추가하여 대뇌 cortices을 소화ypLE 익스프레스. Parafilm와 튜브의 뚜껑을 밀봉하고 10 분간 37 ° C의 물을 욕조에 튜브는 역부족. </li><li> 뚜껑을 열기 전에 70 %의 에탄올과 함께 튜브를 뿌려서 cortices는 튜브의 아래쪽에 정착하고 뜨는을 제거하도록 허용 최대 절전 E. 10 ML을 추가합니다. 이 단계에게 TrypLE Express를 씻어 세 번 반복합니다. 마지막 단계에서 신중하게 모든 미디어를 제거합니다. </li><li> 부드럽게 (4-5 회) 불타는 광택 유리 파스퇴르을 (직경 약 1 ㎜)를 사용 Neurobasal/B27 전체 매체 중 2 ML에서 cortices를 씹다. 거품을 피하기 위해주의하십시오. </li><li> 직경이 작은 유리 멸균 파스퇴르 피펫 (즉, 직경 1/2-3/4 mm 약 피펫)와 다른 4-5 번 반복합니다. 파스퇴르 피펫에게 이것보다 더 작은을 사용하지 마십시오 아니면 세포를 파괴하는 것입니다. </li><li> 정착 (해당되는 경우, 일반적으로 거의)는 조직의 나머지 조각이 소요됩니다. </li><li> 조직의 정착 조각 남겨두고, 새로운 15 ML 관에 상단 단일 세포 현탁액을 전송합니다. 모피Neurobasal/B27 완전한 매체로 10-12 ML에 세포 현탁액을 희석 군터. </li><li> 잘 섞어서 1.5 ML Eppendorf 튜브에 50x 카운팅 솔루션 (표 III 참조) 490 μl에 세포 현탁액의 10 μl를 추가하여 계산을위한 세포를 희석. </li><li> 5.0 × 10 4 / cm 2의 밀도에서 PDL-코팅 차량 번호판 플레이트 세포. nucleofection가 수행되어야하는 경우, 우리는 더 높은 농도 (8-10 × 10 4 / cm 2)에 세포를 도금하는 것이 좋습니다. </li><li> 약 13 X 10 6 뉴런은 각 E17 태아에서 파생됩니다로서 일반적으로, 우리는 실험마다 9-10 fetuses 해부. 더 많은 배아가 필요한 경우, 모든 절차가 2 시간 이상 지속 있지 않은지 확인하십시오. </li><li> 원하는 경우, 24 시간 격리 후, 사이 토신-β-D-arabinofuranoside (AraC) 10 μm의는 glial 확산을 방지하기 위해, 각 요리에 추가할 수 있습니다. Neurobasal/B27 매체가 glial 증식을 억제 이후 그러나,이 단계는 필요하지 않습니다같은 제조 업체의 권고 당 (Invitrogen / Gibco). </li><li> 정확한 시간은 원하는 분화 단계에 따라 달라집니다 있지만 뉴런은 체외에서 4~5일 후 실험에 사용될 수 있습니다. 우리는 생존에 큰 감소 (그림 1)없이 최대 4 주를위한 교양 뉴런 있습니다. </li><li> 확장 culturing 들어, 신선한 Neurobasal/B27 완전한 매체로 매주 culturing 매체를 교체하십시오. </li></ol> 5. 대표 결과 유리 챔버 슬라이드에서 배양해 신경 세포는 immunocytochemistry를 받게됩니다. 그림 1은 문화의 수술 5 일 후에 고정하고의 연결 프로세스를 보여 방지 MAP-2 항체와 immunolabeled 대뇌 피질의 신경 세포의 전형적인 이미지를 보여줍니다. 그림 2는 문화 3 주 후에 쥐 hippocampal 신경 세포의 대표 이미지를 보여줍니다. 완전히 차별화된 세포의 연결 형태는 MAP-2에 의해 강조되는 전mmunolabeling (MAP-2의 연결 마커, 마우스 단클론 항체 클론 AP-20, 텍사스 진, 얼바인, 캘리포니아) 이전에 1에서 설명하는 것처럼 표준 절차에 따라. 이미지 EXI 아쿠아 카메라 (Qimaging), 동력 Z 축 및 SlideBook5 수집 / deconvolution 소프트웨어 (지능형 이미징 이노베이션, 주식, 덴버, CO)가 장착된 니콘 이클립스 E400 직립 형광 현미경으로 시각했다. 각 그림의 입체 영상 시리즈는 한 2 차원 사진에 deconvoluted와 해상도를 높이기 위해 근처 최대한의 강도로 신호를 차단 조정하여 해결이되었습니다. 그림 3의 연결을 문화의 순도를 보여줍니다. 단백질 lysates은 DIV7 쥐의 연결을 문화 (CTX)에서와 인간 glioblastoma (GBM)의 사례에서 얻을되었다. 예상했던대로의 연결 lysate는 단백질의 연결 MAP-2 및 astrocytic 마커 GFAP에 대한 부정에 대한 강한 긍정이다; GBM 단백질 lysate는 부정적인 F 동안또는 GFAP에 대한 MAP-2 및 양성. 우리의 프로토콜에 우리가 매체를 해부하고 rinsing 등 몇 년 동안 최대 절전 모드 E를 사용하고 있습니다만, 최근에 우리는 더 이상 사용하기 위해 뇌 조직을 보존하기 위해 그것을 추가하고 매우 실용적인 사용을 살펴 보았다. 그림 4는 시험 관내 5 (DIV5)의 일 설명 배아에서 원래 절개 후에 최대 절전 E + B27에 1 주일 동안 4 ° C에 보관 cortices 격리 쥐의 대뇌 피질 뉴런의 문화. 뉴런는 이전에 설명한대로 PDL과 laminin과 코팅 유리 두 챔버 슬라이드를 도금했다. 획득한 이미지가 SlideBook5 수집 / 위에서 설명한대로 deconvolution 소프트웨어 (그림 2)를 사용 deconvoluted되었습니다. <img alt="그림 1" src="/files/ftp_upload/3965/3965fig1.jpg" /> 그림 1. pmaxGFP (Amaxa, Lonza, Walkersville, MD)와 nucleofected 및지도 immunolabeled 대뇌 피질의 신경 세포 대표 이미지-2 항체, 빨간색 인치 원본 배율 100x. <img alt="그림 2" src="/files/ftp_upload/3965/3965fig2.jpg" /> 그림 2. 문화 3 주 후에 hippocampal 뉴런 중에 빨간색으로, MAP-2 immunolabeling을 보여주는 대표 이미지입니다. DAPI의 염색법은 파란색으로, 세포의 핵을 보여줍니다. 원본 배율 40x. <img alt="그림 3" src="/files/ftp_upload/3965/3965fig3.jpg" /> 의 연결을 세포 배양의 순도를 보여주는 그림 3. 서양 얼룩. 쥐의 연결과 인간 GBM 단백질 lysates의 30 μg는 전기 영동에 의해 분리 및 표준 절차 1을 다음과 서양 얼룩 분석에 노출되었다. 안티 - MAP-2가 세포 시그널링 (덴버스, MA)에서 polyclonal 토끼, 안티 GFAP 항체는 Chemicon (Millipore, Billerica, MA)에서 단클론 마우스이었고, 마우스 단클론 항 GRB2 항체는 BD의 형질 도입 연구소 (에서왔다 스파크, MD). GRB2은 하중 제어로 사용되었다. </P&gt; <img alt="그림 4" src="/files/ftp_upload/3965/3965fig4.jpg" /> 그림 4. 대표 체외 5 일 사진 (DIV5)에 남아 cortices에서 얻은 쥐의 대뇌 피질의 뉴런 최대 절전 E 그들의 절개 후 일주일 동안 ° C에서 4 번 + B27. 유리 두 챔버 슬라이드에서 배양해 신경 세포의) 위상 대조. 원본 배율 20X. B) Immunofluorescence 녹색에의 연결 과정에 MAP-2의 표현을 보여주는, 문화가 astrocytic 마커 GFAP에 대해 부정적이었다. DAPI의 염색법은 파란색으로, 세포의 핵을 나타냅니다. 원본 배율 40x.

<ol>
	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

쥐 hippocampal과 대뇌 피질 뉴런의 해부 · 문화의 방법은 여기에서 설명한 화학적으로 정의된 매체 (그림 3) 재배 거의 순수의 연결 문화권을 사용하여 실험을 수행할 수 있습니다. 혈청 프리 미디어 culturing 거의 순수 뉴런에 대한 프로토콜 이전에 2,3,4를 설명되어 있지만, 우리의 방법으로 만들어진 중요한 변화가 없습니다. 기존의 프로토콜과는 다른 (즉 은행 외.) 5, 우...

<table border="1"><tbody><tr><td> 시약 </td><td> 집중 </td></tr><tr><td> Neurobasal </td><td> 98% </td></tr><tr><td> B27 </td><td> 2퍼센트 </td></tr><tr><td> Glutamax </td><td> 0.5 MM </td></tr></tbody></table> 표 I. Neurobasal/B27 완료 매체. <table border="1"><tbody><tr><td> 시약 </td><td> 집중 </td></tr><tr><td> 포도당 </td><td> 16 MM </td></tr><tr><td> 자당 </td><td> 22 MM </td></tr><tr><td> HEPES </td><td> 10 MM </td></tr><tr><td> NaCl </td><td> 160 MM </td></tr><tr><td> KCl </td><td> 5 MM </td></tr><tr><td> 나 2 HPO 4 </td><td> 1 ㎜ </td></tr><tr><td> KH 2 PO 2 </td><td> 0.22 MM </td></tr><tr><td> Gentamicin </td><td> 50 μg / ML </td></TR&gt; <tr><td> Fungizone </td><td> 250 NG / ML </td></tr><tr><td> 산도 </td><td> 7.4 </td></tr><tr><td> Osmolarity </td><td> 320-330 mOsm </td></tr></tbody></table> 표 II. 해부 매체. <table border="1"><tbody><tr><td> 시약 </td><td> 볼륨 (μl) </td></tr><tr><td> Neurobasal/B27 완료 매체 </td><td> 240 </td></tr><tr><td> Trypan 블루는 0.4 %를 청바지 </td><td> 250 </td></tr><tr><td> 합계 </td><td> 490 </td></tr></tbody></table> 표 III. 솔루션을 계산 50x. <table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 고양이. 번호 </td></tr><tr><td> 최대 절전 E </td><td> Brainbits </td><td> 767,171 </td></tr><tr><td> Neurobasal </td><td> Gibco, Invitrogen </td><td> 21103-049 </td></tr><tr><td> B27 </td><td> Gibco, Invitrogen </td><td> 17504-044 </td></tr><tr><td> Fungizone </td><td> Gibco, Invitrogen </td><td> 15290-018 </td></tr><tr><td> Gentamicin 황산 </td><td> 시그마 알드리치 </td><td> G1264 </td></tr><tr><td> Glutamax 200 MM </td><td> Gibco, Invitrogen </td><td> 35,050 </td></tr><tr><td> TrypLE 익스프레스 W / O 페놀 레드 </td><td> Gibco, Invitrogen </td><td> 12,604 </td></tr><tr><td> 사이 토신-β-D-arabinofuranoside 하이드로 클로라이드 </td><td> 시그마 알드리치 </td><td> C6645 </td></tr><tr><td> 폴리-D-라이신 </td><td> 시그마 알드리치 </td><td> P6407 </td></tr><tr><td> Laminin 1 MG / ML </td><td> Millipore </td><td> CC095 </td></tr><tr><td> HEPES </td><td> 시그마 알드리치 </td><td> H3375 </td></tr><tr><td> Trypan 블루는 0.4 %를 청바지 </td><td> Gibco, Invitrogen </td><td> 15,250 </td></tr></tbody></테이블&gt; 표 IV. 특정 시약. <table border="1"><tbody><tr><td> 장비 </td><td> 회사 </td><td> 고양이. 번호 </td></tr><tr><td> 스테레오 현미경 </td><td> 올림포스 산 </td><td> SZ61 </td></tr><tr><td> 대형 집게 </td><td> FST </td><td> 11022-14 </td></tr><tr><td> 미세 스쳐 포셉 </td><td> 모리아 </td><td> MC40B </td></tr><tr><td> 마이크로 미세 스쳐 포셉 </td><td> 모리아 </td><td> MC31 </td></tr><tr><td> 면도칼 같은 날카로운 가위 </td><td> Roboz </td><td> RS-6820 </td></tr><tr><td> 마이크로 분석 해 봅시다 가위 </td><td> FST </td><td> 91460-11 </td></tr><tr><td> 마이크로 곡선 가위 분석 해 봅시다 </td><td> FST </td><td> 14067-11 </td></tr><tr><td> 유리 2 챔버 슬라이드 </td><td> 실험실 - 테크 </td><td> 154,461 </td></tr><tr><td> 60mm 요리 </td&gt; <td> BD 팔콘 </td><td> 353,002 </td></tr><tr><td> 100mm 요리 </td><td> 코닝 </td><td> 430,167 </td></tr><tr><td> 15 ML 튜브 </td><td> BD 팔콘 </td><td> 352,099 </td></tr><tr><td> 1.5 ML cryo-튜브 약병 </td><td> Nunc </td><td> 375,353 </td></tr></tbody></table> 표 V. 특정 장비.

isolation and culture of rat embryonic neural cells: a quick protocol

우리는 E15-17 쥐의 배아에서 hippocampal 또는 대뇌 피질의 뉴런을 해리와 문화에 빠른 방법을 설명합니다. 절차는 마우스와 인간의 기본 뉴런과 신경 progenitors의 분리에 성공적으로 적용할 수 있습니다. Dissociated 뉴런은 몇 주간에 혈청이없는 배지에 보관됩니다. 이러한 문화는 nucleofection, immunocytochemistry, 핵산 준비뿐만 아니라, 전기 생리학을 위해 사용될 수 있습니다. 이전의 연결을 문화도 좋은 효율 lentiviral 형질 도입에 의한 속도와, 덜 효율적으로 인산 칼슘이나 lipofectamine과 같은 지질 기반 방법과 함께 transfected 수 있습니다.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

우리는 쥐 배아에서 hippocampal과 대뇌 피질의 신경 세포를 분리하고 문화에 빠른 방법을 설명합니다. 이 프로토콜은 우리가 거​​의 순수의 연결을 문화가 요구되는 실험을 수행할 수 있습니다.

쥐 배아 신경 세포의 분리 및 문화 : 빠른 프로토콜

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

isolation-and-culture-of-rat-embryonic-neural-cells-a-quick-protocol

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JoVE Journal

Neuroscience

37.9K 조회수.  LSU Health Sciences Center - New Orleans. 우리는 쥐 배아에서 hippocampal과 대뇌 피질의 신경 세포를 분리하고 문화에 빠른 방법을 설명합니다. 이 프로토콜은 우리가 거​​의 순수의 연결을 문화가 요구되는 실험을 수행할 수 있습니다.

비디오: Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

연구

신경과학

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

절연 및 Neurosphere 어세를 사용하여 성인 마우스 신경 줄기 세포의 확장

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

인간의 모공에서 신경 크레스트 줄기 세포의 분리와 문화

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

마우스 배아의 Motoneurons의 렉틴 기반 격리와 문화

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

인간 Fungiform의 맛 용의자 전지의 분리 및 문화

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

하나의 마우스, 두 문화 : 분리 및 성인 신경의 문화가 개인 마우스의 두 신경 인성 영역에서 줄기 세포

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...