Name: preparation of rat oligodendrocyte progenitor cultures and quantification of oligodendrogenesis using dual-infrared fluorescence scanning
Uploaded: 2016-02-17T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning at JoVE.com

; 건강의 국립 연구소 : 스폰서 부여 허가 번호 : R37 NS041435.

동물 과목을 포함하는 절차는 기관 동물 관리 및 사용위원회 (IACUC)와 존스 홉킨스 의과 대학에 의해 승인되었습니다. 분석 후판 재고 솔루션 및 OPC 기반 미디어 1. 준비 참고 : 여기에 설명 된 쥐 OPC베이스 (사토) 미디어는 이전에 발표 된 연구 12, 13에서 파생되었습니다. 대안 미디어 제제는이 절차와 호환 할 수있다. <ol><li> 멸균 증류수에 10 ㎍ / ml의 농도로 재현 탁 폴리 -L- 리...

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여기에 제시된 프로토콜은 쥐 개 OPC를 분리하고 실험적인 요인에 반응하여 체외 oligodendrogenesis의 정량화를위한 ​​빠르고 신뢰할 수있는 방법을 설명합니다. 긍정적 인 선택을 사용 가능한 순수 개 OPC의 높은 수율은 실험 목적에 직접 사용됩니다. 이 방법은 1 주일 기간에 분리 배양, 분화 단계를 간소화하고, 고정 된 세포는 편리하게 분석 감도의 손실없이 몇 주 동안 4 ℃에서 저장 될 수?...

포유 동물의 중추 신경계 (CNS)의 효율적인 신경 전도는 희소 돌기 아교 세포에 의한 축삭의 수초가 필요합니다. 개발하는 동안, 희소 돌기 아교 세포는 개발 전뇌 신경 관 (1)의 심실 영역에서 마이그레이션 생각되는 희소 돌기 아교 세포의 전구 세포 (개 OPC)의 풀에서 발생한다. 마이그레이션 개 OPC는 성숙 myelinating 희소 돌기 아교 세포로 분화 한 후 효율적인 임펄스 전파를 용이하게 할뿐만 아니라 영양 지원 2 축삭을 제공 할뿐만 아니라. 성인 CNS는 모든 셀 3의 약 5-8%를 포함하는 회색과 흰색 물질 전반에 걸쳐 분산 개 OPC의 풍부한 인구를 유지한다. 탈수 초성 모욕에 따라, 개 OPC는 부상의 사이트로 마이그레이션 증식, 노출 축삭에 분실 또는 손상된 미엘린 수초를 대체하는 차별화. 그러나, 일부 질병 / 부상 설정에서,이 공정은 비효율적 인 것으로 판명되거나 완전히 4 실패 할 수있다. 동안만성 탈수 초화는 증상 (5)을 완화 할 수있다 질환, 효율적인 oligodendrogenesis 및 재유 수화의 부담에 추가 할 생각된다. 따라서 oligodendrogenesis에 실험 인자의 효과를 결정하기 위해 시험 관내 연구 OPC에 관심있다. 희소 돌기 아교 세포 분화의 상이한 단계로 통찰 단계 특정 세포 마커의 식별에 의해 가능하게되었다. 자기 갱신 초기 전구 세포, 혈소판 유래 성장 인자 수용체 (α)의 발현 (PDGFRα)에 의해 정의되는, 신경 / 신경교 항원이 (NG2)과 A2B5 6-8. OPC를 자신의 분화 과정을 개시하고, 세포주기로부터 탈퇴, 그들은 이러한 마커의 발현을 하향 조절 및 고리 뉴클레오티드 3&#39;- 포스 (CNPase) 및 희소 돌기 아교 세포를 포함 O4 premyelinating 나타내는 단백질을 발현하기 시작한다. 마지막으로, 더 성숙한 oligodendroc으로 분화, 킬로바이트는 미엘린 관련 당 단백질 (MAG), 테오 단백질 (PLP), 및 미엘린 염기성 단백질 (MBP)을 포함하여 수초 관련 단백질의 발현을 특징으로한다. MBP는 세포 내 주변 막 단백질과 수초의 주요 구성 요소입니다. MBP 결핍 마우스는 CNS의 수초 크게 떨림, 경련 및 조기 사망 9,10 선도 저하되는 심각한 표현형을 개발한다. 수초에서 MBP의 중요한 역할은 생체 외 및 생체 (11) 모두 희소 돌기 아교 세포 분화 마커로서의 사용하게되었다. MBP 정량은 몇 가지 다른 방법을 사용하여 달성 될 수있다. RT-PCR 및 웨스턴 블롯 분석은 다른 치료 요법에서 MBP 수준의 정량화를 허용합니다. 면역 세포는 현미경 카메라 기반 방식이 사용되는 경우도있을 수있는 양적 질적 일반적인 접근법이다. 이 시스템은 안정적이고 근본적인 있지만희소 돌기 아교 세포 분화 연구는 이들이 각각의 약물 선별에서 이들의 사용을 제한하는 고유 단점을 갖는다. 우선, RT-PCR 및 웨스턴 블롯 분석을 위해 필요한 기본 OPC에 량이 동시에 검사 할 수있는 변수의 수를 감소시킨다. 면역 세포에 대한 세포의 요구 사항이 훨씬 낮은 반면 정량화가 목표 인 경우, 상당한 시간이 각 실험에 전념해야합니다. 다수의 이미지를 캡쳐 한 후 실험적 분석을 용이하게 정량한다. 이러한주의 사항은 높은 처리량 평가를 필요로 연구에 고려해야 할 중요한 장애물이된다. 크게 필요한 세포의 수 및 정량 분석​​을 완료하는 데 시간을 모두 줄이면서 여기에서 우리는, 면역 세포 및 수초의 정량 웨스턴 블롯 방법론 모두에서 근본적인 측면을 활용하는 방법을 설명합니다.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:691px;">Dissection Materials</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PBS without Ca2+ and Mg2+</td>
			<td>Mediatech</td>
			<td>21-040-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Polystyrene Petri Dishes</td>
			<td>Fisherbrand</td>
			<td>0857512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Dissection Microscope</td>
			<td>Motic</td>
			<td>SM2-168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Dissociation Materials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>Dissociation Tube Rotator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neural Tissue Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td>Enzymatic Dissociation Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#956;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">A2B5 Column Purification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-A2B5 Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-864</td>
			<td>Bead Bound A2B5 Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Magnetic Selection Columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Corning</td>
			<td>46-034-Cl</td>
			<td>2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>0.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDGF-AA</td>
			<td>PeproTech Inc</td>
			<td>100-13A</td>
			<td>20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Hybri-Max 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodothyronine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td>45 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clemastine fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>SML0445-100MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quetiapine hemifumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>Q3638-10MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td>60 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1524</td>
			<td>10 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrecine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>16 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>40 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0135</td>
			<td>50 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">d-Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apo-Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4161</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trace Elements B</td>
			<td>Corning</td>
			<td>25-022-Cl</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Black Visiplate with lid</td>
			<td>Perkin Elmer</td>
			<td>1450-605</td>
			<td>Tissue culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Immunocytochemistry and Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>100-13A</td>
			<td>4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>78787</td>
			<td>0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Jackson Immuno Research</td>
			<td>005-000-121</td>
			<td>5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-MBP</td>
			<td>Biolegend</td>
			<td>SMI-99P</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Actin</td>
			<td>Santa Cruz Biotecnology</td>
			<td>SC-7210</td>
			<td>1:200 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Olig2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse 680RD</td>
			<td>Licor</td>
			<td>926-68070</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit 800CW</td>
			<td>Licor</td>
			<td>926-32211</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-mouse</td>
			<td>Life Technologies</td>
			<td>A11032</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold antifade with DAPI</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Licor Odyssey Clx Infared Imaging System</td>
			<td>Licor</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat oligodendrocyte progenitor cultures and quantification of oligodendrogenesis using dual-infrared fluorescence scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

A2B5 양성 자성는 OPC를 컬럼 분리 시스템을 이용하여 양성 세포의 선택을 통해 분리된다. 분리 과정 이전에, 전체 뇌 P5와 P7 사이에 래트 새끼로부터 제거된다. 전뇌 성공적 두개골로부터 분리되면, 후각 전구 및 소뇌 미세 수술 집게 (도 1A)를 사용하여 제거된다. 대뇌 피질 조직을 분리하기 위해 시상 하부, 시상과 중뇌는 조심 해부 (그림 1B)에 의해...

Watch this Scientific Journal Video about Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning at JoVE.com

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

쥐 희소 돌기 아교 세포 전구 문화의 준비 및 Oligodendrogenesis의 정량화 이중 적외선 형광 스캔을 사용하여

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

출생 후 신경 발생의 연구 Organotypic 슬라이스 문화

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

연구

발생학

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

배달<em&gt; 생체</em&gt; 급성 간헐적 저산소증 신생아 설치류에서 국무 뇌실 지역 유래 신경 전구 세포 배양에

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

뇌실 주위 백질 성인 쥐의 지역 및 인간의 척수에서 신경 줄기 / 전구 세포의 분리

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

약화 된 인간의 진피를 사용하여 유전자 변형의 Organotypic 피부 문화의 생성

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Using Primary Neurosphere Cultures to Study Primary Cilia

차 섬모를 연구에 차 Neurosphere 문화를 사용하여

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

부착 세포 배양에서 시간 경과 형광 현미경 검사 법에 의해 단백질 저하의 단일 셀 정량화

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

내피 콜로니 형성 세포및 중간엽 줄기 세포에 의해 형성된 공동 배양 스페로이드를 이용한 3차원 혈관신생 분석 시스템

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

조혈 줄기 선조 세포 대사 분석

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Quantification of Self-renewal in Murine Mammosphere Cultures

뮤린 맘모스피어 문화에서 자체 갱신의 정량화

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...