이 프로젝트는 캐나다의 여러 경화증 협회에서 RKRWO을 부여하여 투자했던 것은 캐나다의 여러 경화증 협회에서 학생이기의받는 사람입니다. SDR 건강 연구의 캐나다 캐나다 연구소의 다중 경화증 사회에서 포스트 박사 장학금을받는 사람입니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

이 보고서는 OL - 풍부한 문화 또는 DRGN / OL 공동 문화의 차별 화를위한 murine OPCs를 분리하는 방법을 설명합니다. 혼자 교양 경우, OPCs은 MBP로 차별화 + 곳을 OLs을 막의 시트 myelin 같은 생산. neurite 침대 DRGN에 추가하면, OLs은 MBP로 DRGN의 neurites을 싸다 +하신 막합니다. 이 모델은 OL - 중재 axonal ensheathment를 지배하는 복잡한 토대의 조사를 장점. 큰 가치가 있지만, 그러한 문화의 설립은 기술적 도전이다. 특히, 까다로운 측면 효율적인 조직 소화 / 분리, 균형 문화 미디어 산도의 유지 보수 및 DRGN 미디어 변경 사항을 포함합니다. 소화의 길이가, 조직의 금액은 소화 및 분쇄의 금액은 조직 분리의 효능과 최종 결과에 영향을 받고 있다고 의심하는 것이 중요합니다. 그것은 dissociated 신경계 조직에서 낮은 세포 수율을 얻는 경험 연구원 드문되지 않습니다. 또한, murine OPCs 특히 알칼리성 조건, 문화 미디어의 산도 변화에 민감한 경향이 있습니다. 8.5 %의 CO 2에 문화의 유지 보수 OPCs 더 기본 이상의 약산성 조건을 내버려 표시 있기 때문에, 이것을 방지하는 것을 목표로하고있다. 수유 DRGNs와 관련, 미디어 변경은 뉴런을 마르다하지 최대한 빨리 수행되어야하지만, 개발 neurite 침대를 중단하지로 부드럽게해야합니다. 돌발 미디어 변경은 기판에서 neurite 침대와 coverslip로부터 완전한 분리의 가능성이 결과를 이동시키다 수 있습니다. 이 모델 시스템의 잠재적인 장점은 크게는 기술적으로 까다로운 성격을 overshadows. 이 시스템의 장점 중 하나는 배아 조직을 수확하기 위해 번식 여자를 희생의 필요성을 우회, 세포 배양 유래에 대한 사후 산후 마우스를 사용하는 것입니다. 또 다른 장점은 OPCs의 확장에 대한 성장 요인에 대한 요구의 부족 (GFS)입니다. 혼합 glial 문화 astrocyte - 파생 영양 요인의 존재로 인해 아마도 OPCs의 전파를 지원하는 환경을 제공합니다. 이러한 neurospheres 7,8를 통해 유도와 같은 다른 방법은, 이러한 기본적인 fibroblast의 성장 인자 (bFGF), 표피 성장 인자 (EGF)와 OPC 확장에 대한 혈소판 - 유래 성장 인자 (PDGF)로 GFS의 mitogenic 속성에 의존하고 있습니다. 마찬가지로, 출생 후의 (P5 - 10) DRGNs 위해 마우스를 사용하면 신경 성장 인자 (NGF), 배아 DRGNs 9, 10의 체외 생존에 필요한 neurotrophic 요인과 문화 미디어를 보완의 요구 사항을 방지합니다. 그것은 DRGNs 4 교양 때 부정 OLs의 myelinating 용량에 영향을 미친다으로 NGF를 사용하지 않도록 관심을 것입니다. 의 사용 피하 GF를 보충 대규모 사용할 때 이러한 시약은 비용이 증가함에 따라 미디어 또한, 경제적 이점을하고 있습니다. 아마이 문화 모델의 가장 중요한 이점은 따라서 유전자 변형 마우스 라인의 다양한에서 OPCs 및 DRGNs 모두를 파생하는 기회를 제공, 마우스 전용 티슈로부터 파생됩니다. 이것은 모두 DRGN 및 / 또는 myelination에 적용 OPC 특정 속성의 연구 있습니다. 이것은 axons의 OL - 중재 myelination 규제 수용체 / 리간드 상호 작용을 elucidating 특히 중요한 것입니다. 모두에서는,이 기술은 myelination를 기본 분자 신호를 이해하는 방향으로 애플 리케이션에 의한 신경 과학 연구에 관해 훌륭한 가치입니다.

<html><table border="1"><tbody><tr><td> 제품 이름 </td><td> 회사 </td><td> 제품 번호 </td></tr><tr><td> Dulbecco의 수정 이글 중간 (DMEM) </td><td> Multicell </td><td> 319-005 - CL </td></tr><tr><td> 행크의 밸런스드 소금 솔루션 (HBSS) </td><td> Invitrogen </td><td> 14170-112 </td></tr><tr><td> 최소 필수 미디어 (멤) </td><td> GIBCO </td><td> 12360-038 </td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> GIBCO </td><td> 10091-148 </td></tr><tr><td> 페니실린 - 스트렙토 마이신 (펜 / Strep) </td><td> GIBCO </td><td> 15140-122 </td></tr><tr><td> GlutaMAX </td><td> Invitrogen </td><td> 35050-061 </td></tr><tr><td> 폴리 - L - 리신 </td><td> 시그마 - 알드리치 </td><td> P2636 </td></tr><tr><td> 소 혈청 알부민 (BSA) </td><td> 시그마 - 알드리치 </td><td> A4503 </td></tr><tr><td> 인간 merosin 정화 단백질 (LN2) </td><td> Millipore </td><td> CC085 </td></tr><tr><td> 재조합 쥐 ciliary neurotrophic 요인 (CNTF) </td><td> PeproTech </td><td> 450-50 </td></tr><tr><td> L - 티록신 </td><td> Biochemika </td><td> 89,430 </td></tr><tr><td> 홀로그램 트랜스페린 </td><td> 시그마 - 알드리치 </td><td> T0665 </td></tr><tr><td> B27 보충 </td><td> GIBCO </td><td> 0080085 - SA </td></tr><tr><td> 보빈 인슐린 </td><td> 시그마 - 알드리치 </td><td> I6634 </td></tr><tr><td> 3,3 &#39;,5 - Triiodo - L - thyronine </td><td> 시그마 - 알드리치 </td><td> I6634 </td></tr><tr><td> Progesterone </td><td> 시그마 - 알드리치 </td><td> P8783 </td></tr><tr><td> Putrescine </td><td> 시그마 - 알드리치 </td><td> P7505 </td></tr><tr><td> 나트륨 Selenite </td><td> 시그마 - 알드리치 </td><td> S5261 </td></tr><tr><td> 5 - 플루오로 - 2&#39; - deoxyuridine (FuDR) </td><td> 시그마 - 알드리치 </td><td> F0503 </td></tr><tr><td> Papain 솔루션 </td><td> 워싱턴 </td><td> LS003126 </td></tr><tr><td> DNaseI </td><td> ROCHE </td><td> 1010159001 </td></tr><tr><td> L - 시스테인 </td><td> 시그마 - 알드리치 </td><td> C7352 </td></tr><tr><td> 24 잘 조직 문화 요리 </td><td> Cellstar </td><td> 662-160 </td></tr><tr><td> 환기 캡과 T25 - 조직 문화 flasks </td><td> 코닝 </td><td> 430639 </td></tr><tr><td> 10cm 조직 문화 요리 </td><td> 코닝 </td><td> 430167 </td></tr><tr><td> 10cm 페트리 접시 </td><td> 피셔 사이 언티픽 </td><td> 0,875,713 </td></tr><tr><td> Collagenase </td><td> ROCHE </td><td> 103578 </td></tr><tr><td> CellTrics 50 μm의 필터 (옵션) </td><td> PARTEC </td><td> 04-004-2327 </td></tr><tr><td> Myelin 기본 단백질 (MBP) 항체 </td><td> AbD Serotec </td><td> MCA409S </td></tr><tr><td> NG2 항체 </td><td> Millipore </td><td> AB5320 </td></tr><tr><td> Myelin - 관련 당단백질 (마그) 항체 </td><td> Millipore </td><td> MAB1567 </td></tr><tr><td> 2 &#39;, 3&#39; - 순환 - 염기 3&#39; - phosphodiesterase (CNP) 항체 </td><td> Covance </td><td> SMI - 91R - 100 </td></tr><tr><td> 굴지 팬 AB - 5 항체 </td><td> 피츠제럴드 </td><td> 10R - A106AX </td></tr><tr><td> Neurofilament - 200 (NF) 항체 </td><td> 시그마 - 알드리치 </td><td> N4142 </td></tr><tr><td> Tubulin 베타 - 3 체인 (Tuj1) 항체 </td><td> Millipore </td><td> MAB5544 </td></tr><tr><td> 알렉사 플루어 488 염소 안티 - 토끼 IgG (H + L) 이차 항체 </td><td> Invitrogen </td><td> A11008 </td></tr><tr><td> 알렉사 플루어 555 염소 안티 - 마우스 IgG (H + L) 이차 항체 </td><td> Invitrogen </td><td> A21422 </td></tr><tr><td> 알렉사 플루어 647 염소 안티 쥐 (IgG) (H + L) 이차 항체 </td><td> Invitrogen </td><td> A21247 </td></tr><tr><td> 염소 항 마우스 IgG (H + L) - HRP 복합 이차 항체 </td><td> BioRad </td><td> 170-6516 </td></tr><tr><td> 염소 안티 쥐 IgG (H + L) - HRP 복합 이차 항체 </td><td> 산타크루즈 생명 공학 </td><td> SC - 2065 </td></tr><tr><td> 4 &#39;, 6 diamidino - 2 - phenylindole (DAPI) </td><td> 시그마 - 알드리치 </td><td> D9542 </td></tr></tbody></table> 미디어 요리법 100X OL - 보충 * <table border="1"><tbody><tr><td> 성분 </td><td> 추가 금액 </td></tr><tr><td> DMEM </td><td> 100 ML </td></tr><tr><td> BSA </td><td> 1.02 g </td></tr><tr><td> Progesterone </td><td> 0.6 MG </td></tr><tr><td> Putrescine </td><td> 161 MG </td></tr><tr><td> 나트륨 Selenite </td><td> 0.05 MG </td></tr><tr><td> 3,3 &#39;,5 - Triiodo - L - thyronine </td><td> 4 MG </td></tr></tbody></table> -80시 * 보관 ° C 250 μL aliquots에 t &quot;&gt; OL 미디어 <table border="1"><tbody><tr><td> 성분 </td><td> 추가 금액 </td></tr><tr><td> DMEM </td><td> 23.75 ML </td></tr><tr><td> 100X OL - 보충 </td><td> 250 μL </td></tr><tr><td> 보빈 인슐린 (1 MG / ML 주식에서) </td><td> 125 μL </td></tr><tr><td> GlutaMAX </td><td> 250 μL </td></tr><tr><td> 홀로그램 트랜스페린 (33 MG / ML 주식에서) </td><td> 37.5 μL </td></tr><tr><td> B27 보충 </td><td> 500 μL </td></tr><tr><td> FBS </td><td> 125 μL </td></tr><tr><td> CNTF (50 μL / NG 주식에서) </td><td> 25 μL </td></tr></tbody></table> 혼합 glial 문화 미디어 (DMEM에서 만든) <table border="1"><tbody><tr><td> 성분 </td><td> 최종 농도 </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> 펜 / Strep (재고의 0.33 %) </td><td> 33 단위 / ML 페니실린 33 μg / ML 스트렙토 마이신 </td></tr><tr><td> GlutaMAX </td><td> 1퍼센트 </td></tr></tbody></table> DRGN 미디어 (DMEM에서 만든) <table border="1"><tbody><tr><td> 성분 </td><td> 최종 농도 </td></tr><tr><td> FBS </td><td> 10% </td></tr><tr><td> 펜 / Strep (재고의 1 %) </td><td> 100 단위 / ML 페니실린과 100 μg / ML 스트렙토 마이신 </td></tr></tbody></table> 소화 솔루션 요리법 : OPC papain 솔루션 (멤에서 만든) <table border="1"><tbody><tr><td> 성분 </td><td> 최종 농도 </td></tr><tr><td> Papain 솔루션 </td><td> 1.54 MG / ML </td></tr><tr><td> L - 시스테인 </td><td> 360 μg / ML </td></tr><tr><td> DNaseI </td><td> 60 μg / ML </td></tr></tbody></table> DRG papain 용액 (HBSS에서 만든) <table border="1"><tbody><tr><td> 성분 </td><td> 최종 농도 </td></tr><tr><td> Papain </td><td> 1.54 MG / ML </td></tr><tr><td> L - 시스테인 </td><td> 360 μg / ML </td></tr></tbody></table> DRG Collagenase 용액 (HBSS에서 만든) <table border="1"><tbody><tr><td> 성분 </td><td> 최종 농도 </td></tr><tr><td> Collagenase </td><td> 4 MG / ML </td></tr></tbody></table>

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

OL 개발을 기본 분자 메커니즘을 파악하면 OL 생물학 우리의 지식을 발전에만 중요한 것이 아니라, 또한 여러 경화증 (MS)와 같은 demyelinating 질병의 pathogenesis을 이해하는 데 의미가 있습니다. 세포 개발은 일반적으로 기본 세포 배양 모델 공부입니다. 기본 세포 배양은 생체내에 존재하는 외부 변수의 자유로운 제어 환경을 제공하여 주어진 세포 유형의 평가를 용이하게합니다. 쥐에서 파생된 OL 문화가 OL 생물학에 대한 통찰력의 방대한 양의를 제공하는 동안 마우스의 OL 문화를 확립에 유사한 노력은 주요 장애물을 만났되었습니다. 문화 murine 기본 OLs하는 방법을 개발하는 것은 가능한 유전자 변형 마우스 라인을 활용하기 위해 필수적입니다. 설치류 조직에서 OPCs의 추출을위한 여러 방법 neurosphere의 유도, 차동 부착 정화와 1-3 immunopurification 이르기까지, 설명되었습니다. 많은 방법이 성공을 제공하는 동안, 대부분의 광범위한 문화 시간 및 / 또는 비용이 많이 드는 장비 / 시약을 필요로합니다. 이것을 회피하려면, 원래 맥카시 &amp; 데 Vellis 2 설명한 방법의 적응과 murine 조직에서 OPCs을 정화하는 것이 좋습니다. 이 방법은 신생아 쥐 cortices에서 파생된 혼합 glial 문화 물리적으로 분리 OPCs을 포함한다. 그 결과 OL - 풍부한 문화로 차별화된 수있는 정화 OPC 인구입니다. 이러한 접근 방식으로 인해 자사의 비교적 짧은 문화 시간과 성장 요인이나 immunopanning 항체에 대한 불필요한 요구에 호소합니다. 정화 문화 OL 개발의 메커니즘을 탐구하는 것이 유익하는 동안, 그것은 myelin의 쉬스 형성을 평가하기위한 가장 생리학 관련성이 높은 환경을 제공하지 않습니다. 뉴런과 함께 공동 culturing의 OLs은 axons의 OL - 중재 myelination 규제 분자 토대에 대한 통찰력을 빌려 것입니다. 신경 세포 공동 문화 연구 / OL 많은 경우, 지느러미 루트 신경절의 뉴런 (DRGNs)은 선택의 신경 세포 유형적일 수있다. 그들은 추출, 오염 세포의 최소한, 그리고 짙은 neurite 침대 형성 그들의 용이성으로 인해 OLs 공동 문화에 이상적입니다. xenocultures를 myelinating 쥐를 / 마우스를 사용하는 연구가 출판되었습니다 동안 4-6는 그러한 파생하는 방법은 DRGN / OL 포스트 나탈 murine 조직의 공동 문화를 myelinating 것은 설명되지 않았습니다. 우리가 효과적으로 예상 결과의 예제와 함께, 이러한 문화를 생산하는 방법에 대한 자세한 방법을 제시한다. 이러한 방법은 OL 개발 / myelinating 함수에 관련된 질문을 해결하는 데 유용하고, 유용한 도구는 신경 과학 분야에 있습니다.

Watch this Scientific Journal Video about Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues at JoVE.com

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

이 문서는 성숙 oligodendrocytes (OLs)를 생산하는 차별화 기본 문화에 murine oligodendrocyte 전구체 세포 (OPCs)의 풍부한 인구를 유도하는 방법을 설명합니다. 또한,이 보고서는 생산 기술에 대해 설명합니다 murine 마우스 등의 루트 신경절의 뉴런 (DRGNs)의 neurite 침대에 마우스 OPCs을 심는 공동 문화 myelinating.

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

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

derivation-enriched-oligodendrocyte-cultures-oligodendrocyteneuron

Research

JoVE Journal

Neuroscience

36.2K 조회수.  Ottawa Hospital Research Institute. 이 문서는 성숙 oligodendrocytes (OLs)를 생산하는 차별화 기본 문화에 murine oligodendrocyte 전구체 세포 (OPCs)의 풍부한 인구를 유도하는 방법을 설명합니다. 또한,이 보고서는 생산 기술에 대해 설명합니다 murine 마우스 등의 루트 신경절의 뉴런 (DRGNs)의 neurite 침대에 마우스 OPCs을 심는 공동 문화 myelinating.

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

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

고도의 연결을 강화 문화의 생성을위한 두피 조직 블록의 Cryopreservation

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

연구

신경과학

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

E13 생쥐에서 Glial 제한된 엽 성의 전구 물질의 유도

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

인간 태아 뇌에서 전구 - 파생 Oligodendrocyte 문화 시스템

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

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

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

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

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

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

동시 사전 및 사후 신경 Electrophysiological 녹화에서<em&gt; Xenopus</em&gt; 신경 근육 공동 문화

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

단일 셀의 정밀도와의 연결을 공동 문화의 준비

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Organotypic 슬라이스는 문화 희소 돌기 아교 세포 역학 및 수초화을 공부하기

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

다중 전극 배열에 성장의 Organotypic 척수 공동 문화 기능 재생 조사

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

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

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