이 작품은 자연 과학 및 캐나다의 공학 연구 협의회 (보조금 # 249669, 그리고 # 482986)와 심장과 캐나다의 행정 재단 (보조금 # 485508)으로 운용되고 있습니다. 저자는 실험 프로토콜을 개발에서의 도움을 요 세프 엘 하이에크 박사 치 완 감사드립니다.

동물 처리에 관련된 모든 절차는 제도적 지침 (프로토콜 안돼. 20009387)에 따라 토론토 동물 관리위원회의 대학에 의해 승인되었습니다. 다음과 같은 방법은 해당되는 층류 후드에 무균 도구와 기술을 사용하여 수행해야합니다. 아래의 프로토콜 텍스트에서 구문 &quot;EFH-SFM은&quot;표피 성장 인자, 기초 섬유 아세포 성장 인자와 헤파린과 보충 혈청 무료 미디어를 의미합니다. 이 mitogens가 undifferentiated 상태 4 NPCs을 유지하기 때문에 undifferentiated NPCs의 galvanotaxis를 조사 할 때 EFH-SFM이 사용됩니다. 성숙 세포 유형으로 구별하도록 유도 NPCs의 galvanotaxis을 조사 할 때, &quot;FBS-SFM은&quot;1% 태아 소 혈청을 보충 혈청 무료 미디어를 의미합니다. FBS는 성숙한 신경 phenotypes 5로 NPCs의 차별화를 촉진합니다. 1. 신경 전구체의 분리와 문화 (없음) 비디오에 표시 <ol><li> 자궁 경부 전위를 통해 isofluorane과 희생으로 CD1 마우스를 (6~8주 세) 마취시키다. </li><li> 70 % 에탄올에 머리를 끄게하고 날카로운 해부 가위로 동물을 목을 베다. </li><li> 수술 집게로 머리를 누른 상태에서 두개골을 노출 등쪽 표면에 피부를 제거합니다. </li><li> 메스와 더를 사용하지 않습니다. 11 블레이드, mediolateral 축을 따라 정면 굴에서 두개골을 득점하며 rostrocaudal 방향으로 시상 봉합을 따라. </li><li> 멀리이없는 머리에서 정수리 뼈 껍질. 7 곡선 포셉은 피어스로 뇌 조직을 관리하지 말이지. </li><li> 소뇌 아래에서 시작하여 후각 구근으로 발전 뇌 아래에 얇은 주걱을 삽입합니다. 포셉과 장소에 두개골을 누른 상태에서 부드럽게 두개골에서 뇌를 당겨 즉시 얼음처럼 차가운 인공 뇌척수 (아래 레시피를 참조)에 배치합니다. </li><li> 해부 현미경에서 무균 사용해부 가위와 집게는 중간 선을 따라 반으로 뇌에 상처를 냈어. 중간 (컷) 표면이 이상 직면 수 있도록 각 반구를 회전합니다. </li><li> 반구를 선택하고 안쪽 표면이 위쪽으로 향해있는 코퍼스의 callosum (코퍼스의 callosum의 뒤쪽 지역)의 splenium를 찾습니다. </li><li> 피질의 표면에서 dorsoventral 축을 따라 코퍼스 callosum의 splenium에 절개를합니다. </li><li> 측면 심실의 중간과 측면 벽을 노출 할 수있는 후각 망울을 향해 칼에 베인 피질 껍질을 벗기다. </li><li> 등쪽 표면이 위쪽으로 직면 있도록 반구를 회전하고, NPCs는 6 거주 periventricular 지역을 포함하는 노출 안쪽과 측면 벽을 잘라 및 수집 곡선 microscissors을 사용합니다. </li><li> 반복은 다른 반구에 대한 1.8-1.11 단계를 반복합니다. </li><li> 15 CC 관 트립신 솔루션 (아래 레시피를 참조) 7 ML로 분리 된 조직을 피펫, 그리고 37 ° C에서 로커에 관을 배치25 분을위한 보육. </li><li> 5 분에 1,500 rpm으로 튜브를 원심 분리기, 표면에 뜨는을 대기음, 그리고 (아래 레시피를 참조) 트립신 억제제 솔루션 2 ML의 조직을 resuspend. </li><li> 부드럽게 기포를 피하기 위해 조심스럽게 작은 시추공 파스퇴르 피펫 30-50 번과 조직을 씹다. </li><li> 5 분에 1,500 rpm으로 튜브를 원심 분리기, 표면에 뜨는을 대기음, 그리고 펠렛에게 3-5 번 triturating하여 SFM 1-2 ML (아래 레시피를 참조) resuspend. </li><li> 3 분에 1,500 rpm으로 튜브를 원심 분리기, SFM + EFH의의 표면에 뜨는 및 resuspend 1 ML을 대기음. </li><li> SFM + EFH에 μl 당 10 셀의 밀도에서 T25 문화 플라스크에 혈구와 플레이트 세포와 세포 밀도를 살고 계산합니다. </li><li> 문화 NPCs 구성 자유 부동 주요 neurospheres를 얻을 수 그대로 7 일 동안 성장 할 수 있습니다. </li></ol> 2. Galvanotaxis 상공 회의소 준​​비 <ol><li> 더 세 광장 유리를 놓습니다. 1 커버 용지 (22 X 22 X 0.17 mm) 전하루 아침에 6N 염산이 없음 병. </li><li> 다음 날, 광장 노에서 유리 6 직사각형 조각을 (22 × 5 X 0.17 mm) 잘라 다이아몬드 팁 유리 커터를 사용합니다. 한 표지 용지. </li><li> 층류 흐름 후드에 산 세척 광장 전표 및 직사각형 전표를 전송합니다. 조직 문화 수준 autoclaved 물 다음, 70 % 에탄올로 먼저 사각형과 사각형 스트립을 씻고, 그리고 닦아 킴 건조 할 수 있도록 (추가 불임에 대한, 유리는 공기 건조에 허용 될 수 있습니다.) </li><li> 사각형 유리 슬라이드 중 하나 표면의 경계에 진공 그리스를 적용하고, 60mm 플라스틱 배양 접시의 기본으로 봉인. </li><li> 직사각형 유리 스트립 중 하나 표면의 긴 축을 따라 진공 그리스를 적용하고, 중앙 트로프를 생성하기 위해 광장 유리 슬라이드의 반대 가장자리 (서로에 평행되도록)로 밀봉. </li><li> 층류 흐름 후드에 적어도 15 분 동안 방을 UV-소독. </li><li> 폴리-L-라이시의 피펫 250-300 μl2 시간 동안 실온에서 챔버 및 배양의 중심 트로프로 NE. </li><li> 부화 기간이 종료하기 전에 약 15 분 (아래 레시피 참조) Matrigel 솔루션을 준비합니다. </li><li> , 폴리-L-라이신을 기음 autoclaved 물 1 ML, 그리고 중앙 골짜기로 Matrigel 솔루션의 피펫 250-300 μl를 중앙 골짜기을 씻는다. </li><li> 1 시간에 37 ° C에서 방을 품다. </li><li> Matrigel 솔루션을 기음 부드럽게 SFM 1-2 ML과 중앙 골짜기을 씻는다. </li><li> 피펫 100 중앙 골짜기로 EFH-SFM 또는 FBS-SFM의 μl와 계산 현미경의 무대에 galvanotaxis 방을 전송할 수 있습니다. </li><li> 피펫 3-4 60mm 페트리 접시에 neurosphere 함유 문화의 ML하고 계산 현미경의 무대에 페트리 접시를 전송할 수 있습니다. </li><li> 5 배의 볼 목적에서, 각 galvanotaxis의 중심 구유에 5-8 전체 neurospheres을 (최대 4 번) 전송 P10 피펫을 사용하여을 dissociating, 그리고 조심스럽게 Matrigel 기판을 중단하지 않고 중앙 구유 주변의 neurospheres을 전파하지 않고 챔버. </li><li> 중앙 골짜기에 EFH-SFM 또는 FBS-SFM의 추가 150-200 μl를 추가합니다. </li><li> 37 ° C, 5 % CO 2, 17-20 시간 100 % humidified 인큐베이터에 galvanotaxis 챔버를 전송 (있는 경우 undifferentiated NPCs를 분석) 그림 1과 같이 neurospheres이 Matrigel 기판에 준수 및 단일 세포로 해리 할 수 있도록 . 차별화 된 NPCs를 분석하면, 잠복기는 세포의 분화를 허용하도록 69-72 시간까지 연장해야합니다. </li></ol> 3. 셀 시간 경과 영상 라이브 <ol><li> 라이브 셀 이미징 시스템은 37 평형 할 수 있도록 허용 ° C, 5 % 전에 시간 경과 기록의 개시 30 분 최소 CO 2. </li><li> 한쪽 끝에서 1mm 직경 실버 와이어, 코일 두 12cm의 조각을 잘라, 그리고 Clorox B에 배치여과기는 20 분 동안 자세 / AgCl 전극을 형성한다. </li><li> 계산 현미경의 무대에 galvanotaxis 챔버를 전송하고 다음 기준에 따라 라이브 셀 이미징 마이그레이션 분석에 사용 될 챔버 선택 전) neurospheres는 세포가 가지고해야합니다) 단일 셀 및 II에 거의 dissociated해야 작은 - 투 - 노 프로세스와 원형 morphologies은 세포 기관에서 확장됩니다. </li><li> 별도의 정사각형 번호와 함께 층류 후드에 선택한 galvanotaxis 챔버를 전송합니다. 한 유리 커버 슬립 및 진공 그리스. </li><li> 커버가 autoclaved 물 다음, 70 % 에탄올 처음에 빠져 있으며, 커버 슬립의 두 평행 가장자리에 진공 그리스의 스트립을 적용 씻으십시오. </li><li> , 챔버의 중앙 구유에서 문화 미디어를 대기음 후 신속하게 두 개의 평행 한 직사각형 유리 스트립에있는 그리스 스트립의 나머지는, 효율적으로 지붕​​을 만들어 있도록 챔버에 커버 슬립 (그리스 측 아래로 향해)를 배치챔버합니다. </li><li> 모세관 작용을 통해 중앙 구유에 신선한 EFH-SFM 또는 FBS-SFM의 피펫 100 μl. </li><li> 그림 2에 도시 된 바와 같이, 중앙 트로프의 각 끝에 문화 미디어 풀을 위해 국경을 만드는 진공 그리스를 사용하십시오. </li><li> PVC 관의 두 15cm의 조각을 잘라, 그리고주의 깊게 더 거품이 튜브에서 형성되지 보장 튜브에 아가로 오스 솔루션을 주입하는 18 게이지 바늘로 10 CC의 주사기를 사용하고, 젤 5 분 동안 더욱 강화 할 수 있습니다. </li><li> 아가로 오스 겔 튜브, 자세 / AgCl 전극, 문화 미디어 저수지로 사용됩니다 비어있는 60mm 배양 접시 한 ​​쌍의와 함께 라이브 셀 이미징 시스템에 galvanotaxis 챔버를 전송하고 자세 / AgCl 전극을 포함합니다. galvanotaxis 챔버는 37에서 휴식을 할 수 있도록 허용 ° C, 20-30 분에 대한 5 % CO 2 환경입니다. </li><li> 이 기간 동안 시추 구멍으로 2 빈 배양 접시의 뚜껑과 galvanotaxis 챔버의 페트리 접시의 뚜껑을 준비그 그림 3과 같이 드레 멜 또는 이와 유사한 도구로. </li><li> 피펫 각 빈 페트리 접시에 중앙 구유의 양쪽 위에 EFH-SFM 또는 FBS-SFM의 1-1.5 ML, 그리고 SFM 7-8 ML. 각 요리에 galvanotaxis 챔버의 중앙 트로프와 놓으 자세 / AgCl 전극의 각면에 놓으 페트리 접시. 그림 4에 도시 된 아가로 오스 겔 다리로 전기 연속성을 확립 할 수있는 galvanotaxis 챔버와 배양 접시 사이에 다리 격차. </li><li> 전기 전류를 측정 할 수 일련의 전류계로, 외부 전원 공급 장치에 자세 / AgCl 전극을 연결하고 전원 공급 장치를 켜십시오. 직접 중앙 구유에서 전기장의 강도를 측정하는 전압계를 사용하여 원하는 전기장 강도가 (이 실험실에서 수행 assays와 함께 250 뮤직 비디오 / mm의 dcEF 강도를 활용 달성 될 때까지 전원 공급 장치의 출력을 조정 1 1.5 mA 사이의 전류). </li><li> Initia테는 시간 경과에 라이브 셀 이미징 시스템 모듈 및 실험 시간을 원하는 금액 실행 할 수 있습니다. 분석이 완료되면, 표준 immunostaining 분석을위한 4 % paraformaldehyde에서 셀을 수정합니다. </li></ol>

<ol>
	<li>Borgens, R. B., Shi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+histogenesis+from+morphogenesis+in+the+vertebrate+embryo+by+collapse+of+the+transneural+tube+potential.">Uncoupling histogenesis from morphogenesis in the vertebrate embryo by collapse of the transneural tube potential.</a> Dev. Dyn. 203, 456-467 (1995).</li><li>Song, B., Zhao, M., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+cues+regulate+the+orientation+and+frequency+of+cell+division+and+the+rate+of+wound+healing+in+vivo.">Electrical cues regulate the orientation and frequency of cell division and the rate of wound healing in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 13577-13582 (2002).</li><li>Song, B., Zhao, M., Forrester, J., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+regeneration+and+wound+healing+are+stimulated+and+directed+by+an+endogenous+electrical+field+in+vivo.">Nerve regeneration and wound healing are stimulated and directed by an endogenous electrical field in vivo.</a> Journal of Cell Science. 117, 4681-4690 (2004).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+and+population+analyses+demonstrate+that+an+EGF-responsive+mammalian+embryonic+CNS+precursor+is+a+stem+cell.">Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell.</a> Dev. Biol. 175, 1-13 (1996).</li><li>Chiasson, B. J., Tropepe, V., Morshead, C. M., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mammalian+forebrain+ependymal+and+subependymal+cells+demonstrate+proliferative+potential,+but+only+subependymal+cells+have+neural+stem+cell+characteristics.">Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.</a> J. Neurosci. 19, 4462-4471 (1999).</li><li>Erickson, C. A., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+fibroblast+motility+and+orientation+can+be+influenced+by+physiological+electric+fields.">Embryonic fibroblast motility and orientation can be influenced by physiological electric fields.</a> J. Cell Biol. 98, 296-307 (1984).</li><li>Zhao, M., Agius-Fernandez, A., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+and+directed+migration+of+cultured+corneal+epithelial+cells+in+small+electric+fields+are+serum+dependent.">Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent.</a> J. Cell Sci. 109, 1405-1414 (1996).</li><li>Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-Current+Electrical+Field+Guides+Neuronal+Stem/Progenitor+Cell+Migration.">Direct-Current Electrical Field Guides Neuronal Stem/Progenitor Cell Migration.</a> Stem Cells. 26, 2193-2200 (2008).</li><li>Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nat. Protoc. 2, 1479-1489 (2007).</li><li>Huang, C. -. W., Cheng, J. -. Y., Yen, M. -. H., Young, T. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+lung+cancer+cells+in+a+multiple-electric-field+chip.">Electrotaxis of lung cancer cells in a multiple-electric-field chip.</a> Biosen Bioelectron. 24, 3510-3516 (2009).</li><li>SATO, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input-output+relationship+in+galvanotactic+response+of+Dictyostelium+cells.">Input-output relationship in galvanotactic response of Dictyostelium cells.</a> Biosystems. 88, 261-272 (2007).</li><li>Meng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-217 (2011).</li><li>Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids,+EGF+receptors,+and+intracellular+signals+colocalize+and+are+polarized+in+epithelial+cells+moving+directionally+in+a+physiological+electric+field.">Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.</a> The FASEB Journal. , (2002).</li><li>Fang, K., Ionides, E., Oster, G., Nuccitelli, R., Isseroff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+relocalization+and+kinase+activity+are+necessary+for+directional+migration+of+keratinocytes+in+DC+electric+fields.">Epidermal growth factor receptor relocalization and kinase activity are necessary for directional migration of keratinocytes in DC electric fields.</a> J. Cell Sci. 112, 1967-1976 (1999).</li><li>McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Cell+Behavior+Electrically:+Current+Views+and+Future+Potential.">Controlling Cell Behavior Electrically: Current Views and Future Potential.</a> Physiol. Rev. 85, 943-978 (2005).</li><li>Morshead, C. M., Benveniste, P., Iscove, N. N., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+competence+is+a+rare+property+of+neural+stem+cells+that+may+depend+on+genetic+and+epigenetic+alterations.">Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations.</a> Nat. Med. 8, 268-273 (2002).</li><li>Babona-Pilipos, R., Droujinine, I. A., Popovic, M. R., Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Subependymal+Neural+Precursors,+but+Not+Differentiated+Cells,+Undergo+Rapid+Cathodal+Migration+in+the+Presence+of+Direct+Current+Electric+Fields.">Adult Subependymal Neural Precursors, but Not Differentiated Cells, Undergo Rapid Cathodal Migration in the Presence of Direct Current Electric Fields.</a> PLoS ONE. 6, e23808 (2011).</li><li>Deuschl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Randomized+Trial+of+Deep-Brain+Stimulation+for+Parkinson's+Disease.">A Randomized Trial of Deep-Brain Stimulation for Parkinson's Disease.</a> N. Engl. J. Med. 355, 896-908 (2006).</li><li>Stone, S. S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+Entorhinal+Cortex+Promotes+Adult+Neurogenesis+and+Facilitates+Spatial+Memory.">Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory.</a> J. Neurosci. 31, 13469-13484 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 프로토콜은 이전 연구 7-9의 잘 알려진 방법에서 적응되었습니다. Galvanotactic 챔버는 세포 시딩의 구속, 또는 중앙 트로프 10,11의 microfabrication에 대한 CO 2 레이저 절제를 사용하는 잘 별도의 유리의 건설 등 다양한 기법, 다양한을 사용하여 구성 할 수 있습니다. 일부 기술은 다른 것보다 더 힘든거나 비용이 많이 드는 될 수 있습니다. 우리는 일반적으로 대부분의 세포 생...

<table border="1"><tbody><tr><td height="44"> 항목의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td></td><td></td><td></td><td> 신경 전구체 세포 절연 </td></tr><tr><td> 2M NaCl </td><td> 시그마 </td><td> S5886 </td><td> 11.688 g 100 ML DH 2 O에 용해 </td></tr><tr><td> 1M KCl </td><td> 시그마 </td><td> P5405 </td><td> 7.456 g 100 ML DH 2 O에 용해 </td></tr><tr><td> 100 MgCl 2 </td><td> 시그마 </td><td> M2393 </td><td> 20.33 g 100 ML DH 2 O에 용해 </td></tr><tr><td> 155 MM NaHCO 3 </td><td> 시그마 </td><td> S5761 </td><td> 1.302 g 100 ML DH 2 O에 용해 </td></tr><tr><td> 0.5M 포도당 </td><td> 시그마 </td><td> G6152 </td><td> 9.01 g 100에 용해ML DH 2 O </td></tr><tr><td> 108 MM CaCl 2 </td><td> 시그마 </td><td> C7902 </td><td> 1.59 g 100 ML DH 2 O에 용해 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Gibco </td><td> 15,070 </td><td></td></tr><tr><td> 소 췌장의 트립신 </td><td> 시그마 </td><td> T1005 </td><td></td></tr><tr><td> 양 hyaluronidase 고환 </td><td> 시그마 </td><td> H6254 </td><td></td></tr><tr><td> Kynurenic 산 </td><td> 시그마 </td><td> K3375 </td><td></td></tr><tr><td> Ovomucoid 트립신 억제제 </td><td> 워싱턴 </td><td> LS003086 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12100046 </td><td></td></tr><tr><td> F12 </td><td> Invitrogen </td><td> 21700075 </td><td></td></tr><tr><td> 30 % 포도당 </td><td> 시그마 </td><td> G6152 </td><td></td></tr><tr><td> 7.5 % NaHCO 3 </td><td> 시그마 </td><td> S5761 </td><td></td></tr><tr><td> 100 HEPES </td><td> 시그마 </td><td> H3375 </td><td> 23.83 g 100 ML DH 2 O에 용해 </td></tr><tr><td> L-글루타민 </td><td> Gibco </td><td> 25,030 </td><td></td></tr><tr><td> EGF </td><td> Invitrogen </td><td> PMG8041 </td><td> 20 μl 단위로 호르몬 믹스와 나누어지는 1 ML에 Reconstitute. </td></tr><tr><td> FGF </td><td> Invitrogen </td><td> PHG0226 </td><td> 20 μl 단위로 호르몬 믹스와 나누어지는의 0.5 ML의 Reconstitute. </td></tr><tr><td> 헤파린 </td><td> 시그마 </td><td> H3149 </td><td></td></tr><tr><td> APO-트랜스페린 </td><td> R &amp; D 시스템 </td><td> 3188-AT </td><td> 0.1 g 4 ML DH 2 0에 녹 </td></tr><tr><td> Putrescine </td><td> 시그마 </td><td> P7505 </td><td> APO-트랜스페린에 9.61 밀리그램을 수 있도록 분해lution </td></tr><tr><td> 인슐린 </td><td> 시그마 </td><td> I5500 </td><td> 0.1N HCL의 0.5 ML에 25 밀리그램를 해산하고 DH 2 0 3.5 ML에 추가 </td></tr><tr><td> 셀렌 </td><td> 시그마 </td><td> S9133 </td><td></td></tr><tr><td> Progesterone </td><td> 시그마 </td><td> P6149 </td><td></td></tr><tr><td> 표준 해부 도구 </td><td> 정밀 과학 도구 </td><td></td><td></td></tr><tr><td> 해부 현미경 </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td></td><td></td><td></td><td> Galvanotaxis 상공 회의소 준비 </td></tr><tr><td> 광장 유리 커버 슬라이드 </td><td> VWR </td><td> 16,004 </td><td></td></tr><tr><td> 6N 염산 </td><td> VWR </td><td> BDH3204-1 </td><td></td></tr><tr><td> 높은 진공 그리스 </td><td> 다우 코닝 </td><td></td><td></td></tr><tr><td> 60mm 페트리 요리 </td><td> 피셔 과학 </td><td> 0875713A </td><td></td></tr><tr><td> 폴리-L-라이신 </td><td> 시그마 </td><td> P4707 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td> 150 μl 단위로 해동과 나누어지는 </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10082139 </td><td> NPC의 차별화를 유도 경우에만 사용, 그렇지 않으면 SFM 사용 + EFH 문화 미디어는 위에 표시 </td></tr><tr><td> 현미경을 계산 </td><td> 하늘 </td><td> CKX41 </td><td></td></tr><tr><td></td><td></td><td></td><td> 셀 시간 경과 영상 라이브 </td></tr><tr><td> 실버 와이어 </td><td> 알파 Aesar </td><td> 11,434 </td><td></td></tr><tr><td> UltraPure 아가로 오스 </td><td> Invitrogen </td><td> 15510-027 </td><td></td></tr><tr><td> 열 Inactivated FBS </td><td> 시그마 </td><td> 16140071 </td><td></td></tr><tr><td> PVC 관 </td><td> 피셔 과학 </td><td> 80000006 </td><td> 32분의 3 &quot;ID X 32분의 5&quot;OD </td></tr><tr><td> 표백 </td><td> Clorox </td><td></td><td></td></tr><tr><td> 10 CC 주사기 </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 18 게이지 바늘 </td><td> BD </td><td> 305195 </td><td></td></tr><tr><td> 드레 멜 드릴 </td><td> 드레 멜 </td><td> 모델 750 </td><td></td></tr><tr><td> 역 현미경 humidified, incubated 챔버가 장착 </td><td> Zeiss </td><td> Axiovert-200M </td><td></td></tr><tr><td></td><td></td><td></td><td> 요리법 <table border="1"><tbody><tr><td> 항목 </td><td> 음량 </td></tr><tr><td> 2M NaCl </td><td> 6.2 ML </td></tr><tr><td>1M KCl </td><td> 0.5 ML </td></tr><tr><td> 100 MgCl 2 </td><td> 0.32 ML </td></tr><tr><td> 155mM NaHCO 3 </td><td> 16.9 ML </td></tr><tr><td> 1M 포도당 </td><td> 한 ML </td></tr><tr><td> 108 MM CaCl 2 </td><td> 0.09256 ML </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 한 ML </td></tr><tr><td> Autoclaved 물 </td><td> 74 ML </td></tr></tbody></table> 인공 뇌척수 <table border="1"><tbody><tr><td> 항목 </td><td> 볼륨 또는 질량 </td></tr><tr><td> 인공 뇌척수 </td><td> 30 ML </td></tr><tr><td> 소 췌장의 트립신 </td><td> 40 MG </td></tr><tr><td> 양 hyaluronidase 고환 </td><td> 22.8 MG </td></tr><tr><td> Kynurenic 산 </td><td> 5 MG </td></tr></tbody></table> &lt;STRONG&gt; 트립신 솔루션 <table border="1"><tbody><tr><td> 항목 </td><td> 볼륨 또는 질량 </td></tr><tr><td> SFM </td><td> 15 ML </td></tr><tr><td> Ovomucoid 트립신 억제제 </td><td> 10 MG </td></tr></tbody></table> 트립신 억제제 솔루션 <table border="1"><tbody><tr><td> 항목 </td><td> 음량 </td></tr><tr><td> Autoclaved 물 </td><td> 37 ML </td></tr><tr><td> 10X DMEM/F12 </td><td> 10 ML </td></tr><tr><td> 30 % 포도당 </td><td> 2 ML </td></tr><tr><td> 7.5 % NaHCO 3 </td><td> 1.5 ML </td></tr><tr><td> 100 HEPES </td><td> 0.5 ML </td></tr><tr><td> 트랜스페린, Putrescine 솔루션 </td><td> 4 ML </td></tr><tr><td> 25 MG의 인슐린 솔루션 </td><td> 4 ML </td></tr><tr><td> 셀렌 </td><td> 100 &amp; 무; 리터 </td></tr><tr><td> Progesterone </td><td> 100 μl </td></tr></tbody></table> 호르몬 믹스 (-20 ° C 100 ML 총, 상점) <table border="1"><tbody><tr><td> 항목 </td><td> 음량 </td></tr><tr><td> Autoclaved 물 </td><td> 37.5 ML </td></tr><tr><td> 10X DMEM/F12 (3시 1분) </td><td> 5 ML </td></tr><tr><td> 30 % 포도당 </td><td> 한 ML </td></tr><tr><td> 7.5 % NaHCO 3 </td><td> 0.75 ML </td></tr><tr><td> 100 HEPES </td><td> 0.25 ML </td></tr><tr><td> 호르몬 믹스 </td><td> 5 ML </td></tr><tr><td> L-글루타민 </td><td> 0.5 ML </td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> 0.5 ML </td></tr></tbody></table> 혈청 무료 미디어 EFH-SFM은 : EGF의 10 μl, FGF 10 μl, 그리고 헤파린 FBS-SFM의 3.66 μl를 추가합니다 : 0.5 ML FBS를 추가 <ta경 국경 = &quot;1&quot;&gt; <tbody><tr><td> 항목 </td><td> 음량 </td></tr><tr><td> Matrigel </td><td> 150 μl </td></tr><tr><td> SFM </td><td> 3.6 ML </td></tr></tbody></table> Matrigel 솔루션 Matrigel의 나누어지는이 얼음 상자에 위치와 SFM을 혼합하기 전에 점성 액체를 형성 4-5시간 걸쳐 천천히 해동 할 수 있어야합니다. 이 Matrigel 기판의 부드러운 층의 형성을 보장합니다. 천천히 해동하지 않을 경우, 결과 기판 가능성이 세포 이주를 방해, Matrigel의 clumps 포함됩니다. <table border="1"><tbody><tr><td> 항목 </td><td> 볼륨 또는 질량 </td></tr><tr><td> UltraPure 아가로 오스 </td><td> 10 ML ddH 2 0 300 MG </td></tr><tr><td> SFM 열 Inactivated FBS </td><td> 8 ML 2 ML </td></tr></tbody></table> Matrigel솔루션 15 참조 매 관에 2 ML 열 inactivated FBS과 SFM 8 ML을 섞는다. 믹스 10 ML ddH Erlenmeyer 플라스크 2 0 아가로 오스, 그리고 10 초 간격으로 30 초 동안 전자 레인지에서의 열, 각 10 초 간격 후 전자 레인지에서 솔루션을 제거하고 철저하게 혼합 할 수 있도록. 마지막 10 초 전자 렌지 기간 후, 57 ° C의 물을 욕조에있는 SFM / FBS 솔루션과 점포 아가로 오스 솔루션을 섞는다. </td></tr></tbody></table>

a galvanotaxis assay for analysis of neural precursor cell migration kinetics in an externally applied direct current electric field

신경 줄기 및 성인 포유류의 뇌 전구 세포 (이하 칭했다 신경 전구체 세포) (NPCs)의 발견은 neuroregenerative 전략의 개발이 세포 multipotent과 proliferative 속성을 활용하기위한 연구의 몸 시켰습니다. 이러한 전략의 성공을위한 중요한 단계는 외인성 이식 후 병변 사이트 향해 나 CNS의 periventricular 지역에서 발견되는 내생 전구체의 반응을 향상시키기 위해 NPCs의 동원이다. 따라서, 그것은, 홍보 안내 및 NPC 마이그레이션을 강화 메커니즘을 이해하는 것이 필수적입니다. galvanotaxis로 알려진 현상​​ - 우리의 작품은 홍보 할 수 직류 전기 분야의 활용 (dcEFs)와 직접 NPC 마이그레이션에 초점을 맞추고 있습니다. 내생 생리 전기 분야는 정상적인 개발 및 상처 수리하는 동안 세포 이주에 대한 중요한 단서로 기능을 수행합니다. 의 약리 중단axolotl 배아의 횡단 신경 튜브의 잠재력은 심각한 발달 기형 1됩니다. 상처 치유의 맥락에서 상처 각막의 수리 율은 직접 dcEF 2-3의 약리 향상과 혼란에 도시 된 바와 같이, 부상 후 발생하는 상피 상처 잠재력의 크기와 상관되어 있습니다. 우리는 외부 적용 dcEF에 노출되면 성인 subependymal NPCs는 체외에 신속하고 감독 cathodal 마이그레이션을받을 것을 증명하고있다. 이 프로토콜에서 우리는 높은 해상도, 단일 셀 수준에서 직접 전지 본체의 translocation (마이그레이션)의 장기 관찰을위한 간단하고 효과적인 galvanotaxis 분석을 만들기위한 우리 연구실의 기술을 설명합니다. 이 분석은 유전자 변형이나 한방 마우스, 짧은 간섭 RNA, 또는 특정 수용체​​ agonists / antagonists의 사용을 통해 세포 운동성에 dcEF의 도입을 규제 메커니즘을 조사에 적합한 것입니다.

동 분석은 250 MV / mm dcEF의 존재에 undifferentiated NPCs는 음극 대한 높은 이동 및 빠른 galvanotaxis (그림 5A, 영화 1) 전시 것을 보여줍니다. dcEF의 부재에서 세포의 무작위 운동 (그림 5B, 영화 2) 관찰된다. 이 필드 강도에서 undifferentiated NPCs의&gt; 98 %가 그들이 이미징하는 동안 전체 6-8 시간에 대한 마이그레이션, 죽은 세포가 마이그레이션되지 않기 때문에이 사람들이 dcEF의 부재 ?...

Watch this Scientific Journal Video about A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field at JoVE.com

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

이 프로토콜에서 우리는 galvanotaxis 동안 신경 전구체 세포 translocation을 얻은 성인 뇌의 시간 경과 영상을 사용하려면 직류 전기장의 응용 프로그램을 허용 사용자 정의 챔버를 구성하는 방법을 보여줍니다.

외부 응용 직류 전기 분야에 신경 전구체 세포 마이그레이션 속도론 분석을위한 Galvanotaxis 분석

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

a-galvanotaxis-assay-for-analysis-neural-precursor-cell-migration

Research

JoVE Journal

Neuroscience

12.4K 조회수.  University of Toronto. 이 프로토콜에서 우리는 galvanotaxis 동안 신경 전구체 세포 translocation을 얻은 성인 뇌의 시간 경과 영상을 사용하려면 직류 전기장의 응용 프로그램을 허용 사용자 정의 챔버를 구성하는 방법을 보여줍니다.

비디오: A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

연구

신경과학

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

수정 Zigmond 회의소 분석을 사용하여 트렁크 신경 크레스트 세포 마이그레이션 분석

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

전체 마운트 근육 분석에 의한 재현성 마우스 좌골 신경 크러시와 중생의 후속 평가

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

크로스 종 이식에 의한 신경 크레스트 마이 그 레이션과 차별화 분석

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

대뇌 피 질의 로컬 필드 전위, 심전도, 치십시오, 그리고 자유롭게 움직이는 쥐에서 리듬 호흡의 동시 녹음

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

전압에 민감한 염료를 사용하여 뇌 슬라이스의 광자 광자 광기록

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

태아 및 성인 신경 줄기 세포의 고함량 선별 분화 및 성숙 분석- 유래 올리고드로시테 전구체 세포 배양

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...