SS, 킬로바이트 및 유럽위원회 (European Commission) FP7 부여 PIRG08-GA-2010-276989, NEUREX에 교육과 연구의 연방 교육부 (BMBF 01 EO 0803), 및 / 독일 연구 재단 기금 SCHA 1442에 Fazit 재단 대학원 친목에 의해 지원 CS에 3-1 저자는 충돌 재정 이익이 없습니다.

1. 혼합 두피 세포의 분리 및 도금 astrocyte 문화를 혼합 대뇌 피질의 세포 고립 P4 마우스 새끼로 P1을 사용하여 수행 할 수 있습니다. 적절한 astrocyte 밀도를 달성하기 위해이 T75 조직 배양 플라스크에 4 마우스 새끼 cortices를 사용 할 필요가 있습니다. 따라서, 다음과 같은 프로토콜의 볼륨은 4 마우스 새끼를 사용하여 셀 준비를 위해 계산됩니다. <ol><li> 분해 절차를 시작하기 전에, astrocyte 문화 미디어 prewarm 30 ML (DMEM, 고 포도당 + 10 %의 열 inactivated 태아 소 혈청 + 1퍼센트 페니실린 / 스트렙토 마이신, 표 참조) 37 ° C. 37 번 1 시간을위한 세포 배양 등급 물에서 50 μg / ML의 농도 ° CO 2 인큐베이터에서 C의 폴리-D-리신 (PDL)의 20 ML과 문장 한 T75 플라스크입니다. </li><li> 분해 절차에 대해 필요한 모든 시약 및 재료를 준비합니다. 당신이 필요합니다 : 수술 가위, 부드러운 고급 포셉, 평면 팁 포셉, 종이 수건, 폐기물 가방, 70 % 에탄올을얼음에 D이 해부 요리 (3.5 cm 직경은) 2 ML의 HBSS 각이 가득. </li><li> 부드럽게 유지하고 70 %의 에탄올과 새끼 쥐의 머리와 목을 스프레이. 동물은 가위를 사용하여 목 베기에 의해 희생된다. </li><li> 두개골을 표시하는 방법으로 두피를 따라 앞쪽으로 뒤쪽 중간 선 절개를, 수행합니다. </li><li> 목에서 코주의 깊게 두개골을 잘라 요. 두 개의 추가 삭감은 뇌에 더 액세스 할 수 있도록 수행됩니다 : 첫 번째 컷은 후각 전구, 두개골 바닥에서 두개골을 분리 할 수​​있는 소뇌의 하부 다른 하나의 앞쪽에 구성되어 있습니다. </li><li> 평면 팁 집게를 사용하여 두개의 날개가 부드럽게 옆에 이성을 상실하고 뇌는 HBSS로 가득 첫 해부 접시에 제거하고 배치됩니다. 얼음에 다시 접시를 놓고 모든 4 뇌를 수확 계속 진행합니다. </li><li> 분해 절차의 나머지 부분은 stereomicroscope에서 수행됩니다. 첫째, 후각 전구와 소뇌는 고급을 사용하여 제거해부 포셉 (그림 1B). </li><li> , cortices을 검색 고급 집게와 뇌의 뒤쪽 끝을 잡아하기 위해, 반구 사이의 중간 선 절개을 수행 생성 된 숲에 포셉의 두 번째 세트를 삽입하고 벗겨 버릴에서 피질의 판과 같은 구조 뇌. </li><li> 조심스럽게 고급 집게 (그림 1D &#39;)로 당겨 피질의 뇌에서 meninges를 해부. 이 단계는 수막 세포와 섬유 아세포에 의한 최종 astrocyte 문화의 오염을 방지합니다. HBSS로 가득 두 번째 접시에 준비한 피질의 뇌를 전송하고 얼음으로 그것을 반환합니다. 모든 4 cortices에 따라 계속합니다. </li><li> 마지막으로, 날카로운 블레이드를 (약 4 8 배)를 사용하여 작은 조각으로 각 피질 반구를 잘라. </li><li> 무균 조건 하에서, 전송 피질하다 50 ML 팔콘 튜브에 조각과 22.5 ML의 총 볼륨에 HBSS를 추가합니다. </li><li> 2.5 %의 트립신, 혼합 2.5 ML를 추가하고 품다37 번 물 목욕의 조직 ° C 30 분에. 매 10 분을 흔들어 가끔하여 섞는다. </li><li> 펠렛 피질 조직 조각 300 XG에서 5 분 동안 원심 분리기. </li><li> 조심스럽게 decantation에 의해 표면에 뜨는 제거합니다. 조직 펠렛을 잃지 않도록하기 위해 당신은 기계적으로는 pipet을 사용하여 유지 될 수 있습니다. 조직 조각이 하나의 세포 (20-30 회)에 dissociated 때까지 10 ML 플라스틱 피펫을 사용하여 10 ML astrocyte 도금 매체와 적극적으로 pipetting을 추가하여 단​​일 세포 현탁액으로 조직을 떼어 놓다. astrocyte 도금 매체를 사용하여 20 ML에 볼륨을 조정합니다. 당신은 hematocytometer를 사용하여 계산하여 단일 세포로 증명 피질 조직의 분리를 할 수 있습니다. 4 마우스 새끼 cortices 중 하나 준비 10-15 X10 6 dissociated 하나의 세포를 얻을 수 있습니다. </li><li> T75 문화 플라스크에서 PDL를 대기음, 판 37 번 dissociated 단일 세포 현탁액 및 배양 ° CO 2 인큐베이터에서 C. </li></ol> 2. Enr 구하기iched Astrocyte 문화 <ol><li> 이후 혼합 대뇌 피질의 세포 및 모든 3 일 도금 후 매체 2 일 변경합니다. </li><li> astrocytes가 합류하고 오버레이 microglia하는 astrocyte 레이어에 노출 앉거나 이미 astrocyte 레이어 (그림 2)에서 떨어 뜨려 7~8일, 후, microglia를 제거 할 수 궤도 셰이커 30 분 동안 180 rpm으로 T75 플라스크를 흔들. 표면에 뜨는 포함 microglia를 삭제 하시겠습니까이나 문화에 희망과 microglia를 검토하는 경우를 스핀 다운과 문화 28,29위한 판. </li><li> 20 ML 신선한 astrocyte 문화 매체를 추가하고 oligodendrocyte 전구체 세포 (OPC)를 제거하기 위해 6 시간에 240 rpm으로 술병을 흔들어하여 계속 진행합니다. 일부 OPCs 완전히 astrocyte 레이어에서 분리되지 않습니다 때문에, OPC 오염을 방지하기 위해 1 분에 손으로 힘차게 흔들하고 있습니다. 표면에 뜨는을 취소하거나 문화에 OPCs 29 원하는 경우, 내려와 플레이트 스핀. </li><li> 나머지 conflue을 씻어PBS로 두 번 NT astrocyte 층은, 37의 CO 2 인큐베이터에서 5 ML 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 및 배양을 추가 ° C., PBS를 대기음 astrocytes의 분리마다 5 분을 확인하고 손 (2-3 번)의 손바닥에 대해 플라스크를 클릭하여 astrocytes의 부대를 시행합니다. </li><li> astrocytes는 문화 플라스크에서 분리 된 후, astrocyte 문화 매체, 5 분 180 XG에서 스핀 세포의 5 ML을 추가 표면에 뜨는을 대기음 40 ML 신선한 astrocyte 도금 매체를 추가 할 수 있습니다. 한 T75 조직 배양 플라스크는 첫 번째 셀 분할 후 6 1x10 주변의 세포를 얻을 수 있습니다. 37 두 개의 T75 문화 플라스크 및 배양에 판 세포 ° CO 2 인큐베이터에서 C. 모든 2~3일 매체를 변경합니다. </li><li> 첫 번째 분할 astrocytes 후 12-14일는 실험을 수행하기 전에 해당 셀 농도 24-48 시간에 도금되어 있습니다. 한 T75 조직 배양 플라스크는 두 번째 셀 분할 후 1.5-2 주변 X10 6 세포를 얻을 수 있습니다. </li></ol>

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	<li>Nedergaard, M., Ransom, B., Goldman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+roles+for+astrocytes:+redefining+the+functional+architecture+of+the+brain.">New roles for astrocytes: redefining the functional architecture of the brain.</a> Trends Neurosci. 26, 523-530 (2003).</li><li>Belanger, M., Allaman, I., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+energy+metabolism:+focus+on+astrocyte-neuron+metabolic+cooperation.">Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation.</a> Cell Metab. 14, 724-738 (2011).</li><li>Allen, N. J., 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=Neuroscience:+Glia+-+more+than+just+brain+glue.">Neuroscience: Glia - more than just brain glue.</a> Nature. 457, 675-677 (2009).</li><li>Ballas, N., Lioy, D. T., Grunseich, C., Mandel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell+autonomous+influence+of+MeCP2-deficient+glia+on+neuronal+dendritic+morphology.">Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology.</a> Nat. Neurosci. 12, 311-317 (2009).</li><li>Jacobs, S., Nathwani, M., Doering, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fragile+X+astrocytes+induce+developmental+delays+in+dendrite+maturation+and+synaptic+protein+expression.">Fragile X astrocytes induce developmental delays in dendrite maturation and synaptic protein expression.</a> BMC Neurosci. 11, 132 (2010).</li><li>Kaneko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+clear+the+path+of+astrocytic+processes+for+their+rapid+migration+in+the+adult+brain.">New neurons clear the path of astrocytic processes for their rapid migration in the adult brain.</a> Neuron. 67, 213-223 (2010).</li><li>Min, R., Nevian, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+signaling+controls+spike+timing-dependent+depression+at+neocortical+synapses.">Astrocyte signaling controls spike timing-dependent depression at neocortical synapses.</a> Nat. Neurosci. , (2012).</li><li>Eroglu, C., 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=Regulation+of+synaptic+connectivity+by+glia.">Regulation of synaptic connectivity by glia.</a> Nature. 468, 223-231 (2010).</li><li>Sasaki, T., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action-potential+modulation+during+axonal+conduction.">Action-potential modulation during axonal conduction.</a> Science. 331, 599-601 (2011).</li><li>Bozoyan, L., Khlghatyan, J., Saghatelyan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+control+the+development+of+the+migration-promoting+vasculature+scaffold+in+the+postnatal+brain+via+VEGF+signaling.">Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling.</a> J. Neurosci. 32, 1687-1704 .</li><li>Alvarez, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hedgehog+pathway+promotes+blood-brain+barrier+integrity+and+CNS+immune+quiescence.">The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence.</a> Science. 334, 1727-1731 (2011).</li><li>Abbott, N. J., Ronnback, L., Hansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-endothelial+interactions+at+the+blood-brain+barrier.">Astrocyte-endothelial interactions at the blood-brain barrier.</a> Nat. Rev. Neurosci. 7, 41-53 (2006).</li><li>Tao-Cheng, J. H., Nagy, Z., Brightman, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions+of+brain+endothelium+in+vitro+are+enhanced+by+astroglia.">Tight junctions of brain endothelium in vitro are enhanced by astroglia.</a> J. Neurosci. 7, 3293-3299 (1987).</li><li>Gordon, G. R., Choi, H. B., Rungta, R. L., Ellis-Davies, G. C., MacVicar, 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=Brain+metabolism+dictates+the+polarity+of+astrocyte+control+over+arterioles.">Brain metabolism dictates the polarity of astrocyte control over arterioles.</a> Nature. 456, 745-749 (2008).</li><li>Silver, 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=Regeneration+beyond+the+glial+scar.">Regeneration beyond the glial scar.</a> Nat. Rev. Neurosci. 5, 146-156 (2004).</li><li>Schachtrup, C., Moan, N. L. e., Passino, M. A., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells+and+astrocytes:+Stars+of+scar+formation+and+tissue+repair.">Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair.</a> Cell Cycle. 10, 1764-1771 (2011).</li><li>Schachtrup, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinogen+triggers+astrocyte+scar+formation+by+promoting+the+availability+of+active+TGF-beta+after+vascular+damage.">Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage.</a> J. Neurosci. 30, 5843-5854 (2010).</li><li>Bushong, E. A., Martone, M. E., Jones, Y. Z., Ellisman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasmic+astrocytes+in+CA1+stratum+radiatum+occupy+separate+anatomical+domains.">Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains.</a> J. Neurosci. 22, 183-192 (2002).</li><li>Ogata, K., Kosaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+quantitative+analysis+of+astrocytes+in+the+mouse+hippocampus.">Structural and quantitative analysis of astrocytes in the mouse hippocampus.</a> Neuroscience. 113, 221-233 (2002).</li><li>Dabir, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+glutamate+transport+in+a+mouse+model+of+tau+pathology+in+astrocytes.">Impaired glutamate transport in a mouse model of tau pathology in astrocytes.</a> J. Neuroscience. 26, 644-654 (2006).</li><li>Wisniewski, H. M., Wegiel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+relationships+between+astrocytes+and+classical+plaque+components.">Spatial relationships between astrocytes and classical plaque components.</a> Neurobiol. Aging. 12, 593-600 (1991).</li><li>Shin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mutant+huntingtin+in+glial+cells+contributes+to+neuronal+excitotoxicity.">Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity.</a> J. Cell Biol. 171, 1001-1012 (2005).</li><li>Wakabayashi, K., Hayashi, S., Yoshimoto, M., Kudo, H., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NACP/alpha-synuclein-positive+filamentous+inclusions+in+astrocytes+and+oligodendrocytes+of+Parkinson's+disease+brains.">NACP/alpha-synuclein-positive filamentous inclusions in astrocytes and oligodendrocytes of Parkinson's disease brains.</a> Acta Neuropathol. 99, 14-20 (2000).</li><li>Lioy, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+glia+in+the+progression+of+Rett's+syndrome.">A role for glia in the progression of Rett's syndrome.</a> Nature. 475, 497-500 (2011).</li><li>Quinlan, R. A., Brenner, M., Goldman, J. E., Messing, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP+and+its+role+in+Alexander+disease.">GFAP and its role in Alexander disease.</a> Exp. Cell Res. 313, 2077-2087 (2007).</li><li>Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+damage+in+the+central+nervous+system:+a+multifaceted+role+for+vascular-derived+TGF-beta.">Vascular damage in the central nervous system: a multifaceted role for vascular-derived TGF-beta.</a> Cell Tissue Res. 347, 187-201 (2012).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J. Cell Biol. 85, 890-902 (1980).</li><li>Siao, C. J., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+plasminogen+activator+mediates+microglial+activation+via+its+finger+domain+through+annexin+II.">Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II.</a> J. Neurosci. 22, 3352-3358 (2002).</li><li>Armstrong, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+immature+oligodendrocyte+lineage+cells.">Isolation and characterization of immature oligodendrocyte lineage cells.</a> Methods. 16, 282-292 (1998).</li><li>Cahoy, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptome+database+for+astrocytes,+neurons,+and+oligodendrocytes:+a+new+resource+for+understanding+brain+development+and+function.">A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.</a> J. Neurosci. 28, 264-278 (2008).</li><li>Anthony, T. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+folate+metabolic+enzyme+ALDH1L1+is+restricted+to+the+midline+of+the+early+CNS,+suggesting+a+role+in+human+neural+tube+defects.">The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects.</a> J. Comp. Neurol. 500, 368-383 (2007).</li><li>Skoff, R. P., Knapp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+astroblasts+and+oligodendroblasts+in+postnatal+rodent+brain:+evidence+for+separate+astrocyte+and+oligodendrocyte+lineages.">Division of astroblasts and oligodendroblasts in postnatal rodent brain: evidence for separate astrocyte and oligodendrocyte lineages.</a> Glia. 4, 165-174 (1991).</li><li>Molofsky, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+and+disease:+a+neurodevelopmental+perspective.">Astrocytes and disease: a neurodevelopmental perspective.</a> Genes Dev. 26, 891-907 (2012).</li><li>Zhang, Y., 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=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr. Opin. Neurobiol. 20, 588-594 (2010).</li><li>Foo, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71, 799-811 (2011).</li><li>Jungblut, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+living+primary+astroglial+cells+using+the+new+GLAST-specific+monoclonal+antibody+ACSA-1.">Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.</a> Glia. 60, 894-907 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 설명 된 방법은 원래 1980 27 맥카시와 드 Vellis에 의해 설명 쥐 신생아 뇌에서 astrocyte 문화 준비에 기반을두고 있습니다. 출생 후의 P1에서 P4 마우스 뇌 피질 astrocytes의 고립과 문화의 수정 방법은 여기에 제시된 것은 수율 순수 기본 astrocytes 빠르고 높은 재현합니다. 이 기술은 쉽게 이러한 쥐 또는 돼지에서와 같은 척수와 같은 다른 뇌 영역에서 같은 다른 종에서 astrocytes을 분리 양?...

Astrocytes는 중추 신경계 (CNS)의 매우 풍부한 세포 유형입니다. 인간의 대뇌 피질 1 뉴런 당 1.4 astrocytes가 반면 뉴런에 astrocytes의 비율은, 쥐와 쥐의 대뇌 피질에 1시 3분입니다. astrocyte 기능에 대한 관심은 최근 몇 년 동안 크게 증가했다. astrocytes의 주요 기능은 뉴런 2,3에 구조와 신진 대사 지원을 제공하는 역할입니다. astrocytes에 새로 발견 된 역할은 기능의 폭 넓은 스펙트럼을 커버. 이러한 개발 4-6 동안 개발 axons과 특정 neuroblasts의 마이그레이션 가이드, 신경 전달의 기능, 신경 회로 7-9으로 시냅스의 강도와 정보 처리, 혈액 - 뇌 장벽의 역할 (BBB)이 형성 10 무결성 11-13을 포함 뇌 혈관 톤 (14)과 규제. astrocytes의 또 다른 주요 기능은 부상에 대한 그들의 반응이다. 병적 인 조건 astrocyt 아래에스 반응이되고 더 중간 필라멘트 glial fibrillary 산성 단백질 (GFAP)와 억제 세포 외 기질 (ECM) 단백질 15,16의 표현을 upregulate. 반응 astrocytes은 콘드로이틴 황산 proteoglycan (CSPG) 가족의 astrocyte 분비 ECM 단백질의 주요 구성 glial 흉터를 형성하여 건강한 조직에서 부상 사이트를 한계를 정하다, 그 주요 요인은 CNS 손상 15-17 후 axonal 재생을 억제. Astrocytes 늦게 embryogenesis과 이른 출생 후의 생활 동안 레이디 얼 glial (RG) 세포에서 발생. astrocyte 사양가 발생했습니다 후 astrocyte 전구체는 최종 위치로 이전, 그들이 터미널 차별화의 과정을 어디서부터 시작. 생체에서 astrocytes는 전형적인 형태 18,19로 표시로 3-4주 출생 후 성인 것 같습니다. RG 세포의 subpopulation은 subventricular 영역 astrocytes (타입 B 세포)로 변환합니다. Both, RG 입력합니다 B 세포는 각각 개발 중에 있으며 성인의 astrocyte 같은 신경 줄기 세포 (NSCs)로 기능을 수행합니다. astrocytes, RG 입력합니다 B 세포와 마찬가지로 또한이 마커가 독점적으로 특별히 성인 astrocytes 라벨을 사용할 수 없습니다 나타냅니다 astrocyte 특정 glutamate의 전송 (GLAST), 뇌 지질 결합 단백질 (BLBP) 및 GFAP를 표현한다. 건강한 뇌, RG 및 자기 갱신에 대한 용량과 같은 타입 B 세포 전시 줄기 세포의 잠재력에 나누어하지 않는 성인 parenchymal astrocytes에 대비합니다. astrocytes의 Dysregulation는 알츠하이머 병 20,21, 헌팅턴 병 22, 파킨슨 병 23, Rett 증후군 24 알렉산더 병 25를 포함하여 많은 pathologies에 연루되었습니다. 또한, astrocytes는 astrocyte 활성화 및 astrocytic glial 흉터 형성 16,26로 이어지는, CNS의 모든 모욕에 반응. 다음 뇌 TR을 형성 astrocytic glial 흉터auma 또는 척수의 부상은 neuronal 재생 15을 방지하는 소수의 장벽이 될 것으로 생각됩니다. 세포의 정화 인구를 분리하고 유지하기 위해 신뢰할 수있는 방법의 개발은 신경​​계에 대한 우리의 이해에 필수적이었습니다. 맥카시와 드 Vellis의 개척 작업은 신생아 쥐 조직 27 일부터 astrocytes의 거의 순수한 문화를 준비하는 날짜로 조사 할 수 있습니다. 대부분 마우스 대뇌 피질의 astrocytes를 격리시킨 것에 대한 약간 수정 형태로 여기에 제공됩니다이 방법을 사용 astrocyte 생물학에 대해 알게되었습니다. 생체 연구, astrocytes뿐만 아니라 설치된 매체가 얻은 체외 문화에 설명을 사용하여 보완, 추가 astrocyte 함수로 정보를 얻으려면 유용한 도구입니다.

<table border="1"><tbody><tr><td> 작업 솔루션의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 최종 농도 </td></tr><tr><td></td><td></td><td></td><td> Astrocyte 문화 미디어 </td></tr><tr><td> DMEM, 높은 혈당 </td><td> 생명 기술 </td><td> 31966-021 </td><td></td></tr><tr><td> 열 inactivated FBS, </td><td> 생명 기술 </td><td> 10082-1​​47 </td><td> 최종 농도 : 10 % </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 생명 기술 </td><td> 15140-122 </td><td> 최종 농도 : 1 % </td></tr><tr><td></td><td></td><td></td><td> 뇌 세포 발굴을위한 솔루션estion </td></tr><tr><td> HBSS </td><td> 생명 기술 </td><td> 14170-088 </td><td></td></tr><tr><td> 2.5 %의 트립신 </td><td> 생명 기술 </td><td> 15090-046 </td><td> 최종 농도 : 0.25 % </td></tr><tr><td></td><td></td><td></td><td> 다른 </td></tr><tr><td> 70 % (권 / 권) 에탄올 </td><td> 로스 </td><td> 9065.2 </td><td></td></tr><tr><td> 폴리-D-라이신 </td><td> Millipore </td><td> A-003-E </td><td> 50 μg / ML </td></tr><tr><td> 물 </td><td> PAA </td><td> S15-012 </td><td> 세포 배양 등급 </td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td> 세포 배양 등급 </td></tr><tr><td> 0.05 %가보세요psin-EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 생명 기술 </td><td> 25300-062 </td><td></td></tr><tr><td> 0.45 μm 무균 필터 </td><td> Sartorius </td><td> 16,555 </td><td></td></tr><tr><td> 3.5 cm 페트리 접시 </td><td> BD 팔콘 </td><td> 353001 </td><td></td></tr><tr><td> 15 ML 팔콘 튜브 </td><td> BD 팔콘 </td><td> 352096 </td><td></td></tr><tr><td> 50 ML 팔콘 튜브 </td><td> BD 팔콘 </td><td> 352070 </td><td></td></tr><tr><td> 75cm 두 조직 문화 플라스크 </td><td> BD 팔콘 </td><td> 353136 </td><td></td></tr><tr><td> 집게, 괜찮아 </td><td> 뒤몽 </td><td> 2-1032, 2-1033 </td><td> # 3C, # 5 </td></tr><tr><td> 집게, 평면 팁 </td><td> KLS 마틴 </td><td> 12-120-11 </td><td></td></tr><tr><td> 13cm 외과 가위 </td><td> Aesculap </td><td> BC-140-R </td><td></td></tr><tr><td> Stereomicroscope </td><td> Leica </td><td></td><td> MZ7.5 </td></tr><tr><td> Stereomicroscope + 카메라 </td><td> Leica </td><td></td><td> MZ16F, DFC320 </td></tr><tr><td> 현미경 + 카메라 </td><td> Zeiss, 캐논 </td><td></td><td> 프리모 초록, 파워의 A650입니다 </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf </td><td> 5805000.017 </td><td> Centrifuge5804R </td></tr><tr><td> 궤도 쉐이커 </td><td> 열 과학 </td><td> SHKE 4450 - 1CE </td><td> MaxQ 4450 </td></tr><tr><td> 열탕 목욕 </td><td> Julabo </td><td></td><td> 에스W20, 37 ° C </td></tr></tbody></table>

isolation and culture of mouse cortical astrocytes

Astrocytes는 포유류의 뇌에 풍부한 세포 유형입니다, 아직 많은 자신의 분자와 기능 특성에 대해 학습 할 수 남아 있습니다. 체외 astrocyte 세포 배양 시스템에 자세히 이러한 glial 세포의 생물학적 기능을 연구하는 데 사용할 수 있습니다. 이 비디오 프로토콜은 마우스 새끼의 혼합 대뇌 피질 세포의 절연 및 문화 순수한 astrocytes를 얻는 방법을 보여줍니다. 방법은 가능한 뉴런의 부재와 astrocytes, oligodendrocytes 및 microglia, 문화의 중추 신경 시스템의 세 가지 주요 glial 세포 인구의 분리에 기반을두고 있습니다. 문화의 첫 번째 일 동안 대표 이미지가 혼합 셀 인구의 존재를 입증하고 astrocytes가 합류되고 microglia 및 oligodendrocytes에서 분리해야 할 때, timepoint을 나타냅니다. 또한, 우리는 순결하고 잘 설립을위한 immunocytochemical stainings를 사용하여 양식 astrocytes의 astrocytic 형태를 보여와새로 astrocyte 마커를 설명했다. 이 문화 시스템은 쉽게 astrocyte 생물학의 다양한 측면을 연구에 대한 순수한 마우스 astrocytes와 astrocyte이 설치된 미디어를 얻을하는 데 사용할 수 있습니다.

전체 마우스의 뇌 (그림 1A), 소뇌 및 후각 구근은 (그림 1B) 제거 할 필요가의 고립시. cortices은 마우스 뇌 줄기 (그림 1C) 및 개인 피질 (그림 1D &#39;)의 meninges의 껍질을 벗겨 아르주의 (그림 1E) 제거됩니다. Meninges는 수막 동맥 시스템 및 수막 세포와 섬유 아세포에 의한 최종 astrocyte 문화의 오염의 불완전 제거 결과로 분명합니다. <p c...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Cortical Astrocytes at JoVE.com

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes 정상 뇌 개발 및 기능, 및 중앙 신경계 수리를 위해 필수적인 기초 생물학적 과정에 참여하는 다양한 세포로 인정되었습니다. 여기 중추 신경계 세포의 주요 클래스의 생물학을 연구하는 순수 마우스 astrocyte 문화를 얻기 위해 빠른 절차를 제시한다.

마우스 두피 Astrocytes의 분리와 문화

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

isolation-and-culture-of-mouse-cortical-astrocytes

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90.8K 조회수.  University of Freiburg. Astrocytes 정상 뇌 개발 및 기능, 및 중앙 신경계 수리를 위해 필수적인 기초 생물학적 과정에 참여하는 다양한 세포로 인정되었습니다. 여기 중추 신경계 세포의 주요 클래스의 생물학을 연구하는 순수 마우스 astrocyte 문화를 얻기 위해 빠른 절차를 제시한다.

비디오: Isolation and Culture of Mouse Cortical Astrocytes

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

연구

신경과학

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. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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 ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

모델링 성상 세포종 병인<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 대뇌 피질의 성상이나 조건, 유전자 조작 쥐에서 신경 줄기 세포를 사용하여

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

마우스 해마 성상에서 섹스 차이점 후<em&gt; 인 체외</em&gt; 허혈

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

대뇌 피 질의 Interneuron 선구자로 마우스 배아 줄기 세포의 분화

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.