Name: isolation of brain and spinal cord mononuclear cells using percoll gradients
Uploaded: 2011-02-02T17:00:00
Description: Watch this Scientific Journal Video about Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients at JoVE.com

这项工作是由国家多发性硬化症协会（TA 3021 - A1 /吨的AEC）和圣安东尼奥的得克萨斯大学的支持。

试剂的制备准备与10X的HBSS部分没有搅拌约9 percoll部分股票等渗percoll（SIP），4毫升，每大脑+ +和Mg + +。 准备在70％1X的HBSS SIP的无钙+ +和Mg + +中，大脑每2毫升到15毫升的聚丙烯锥形管处方。 注：建议percoll应在室温下使用，如果使用的冷，细胞易于聚集和细胞分离效率较低。 组织收集和同质化通?...

<ol>
	<li>Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+direct+characterization+of+resident+microglial+cells+from+the+normal+and+inflamed+central+nervous+system.">Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.</a> Proceedings of the National Academy of Sciences of the United States of America. 88, 7438-7442 (1991).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigen+presentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared.</a> J Immunol. 154, 4309-4321 (1995).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Juedes, A. E., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+infiltrating+central+nervous+system+APCs+regulate+the+emergence+and+resolution+of+experimental+autoimmune+encephalomyelitis.">Resident and infiltrating central nervous system APCs regulate the emergence and resolution of experimental autoimmune encephalomyelitis.</a> J. Immunol. 166, 5168-5175 (2001).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tickets+to+the+brain:+Role+of+CCR2+and+CX(3)CR1+in+myeloid+cell+entry+in+the+CNS.">Tickets to the brain: Role of CCR2 and CX(3)CR1 in myeloid cell entry in the CNS.</a> J Neuroimmunol. , (2010).</li><li>Mildner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat. Neurosci. 10, 1544-1553 (2007).</li><li>Djukic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+monocytes+engraft+in+the+brain,+differentiate+into+microglia+and+contribute+to+the+pathology+following+meningitis+in+mice.">Circulating monocytes engraft in the brain, differentiate into microglia and contribute to the pathology following meningitis in mice.</a> Brain. 129, 2394-2403 (2006).</li></ol>

中枢神经系统的白细胞从正常和炎症鼠标组织中的细胞表面标记的分析已动用1-3几十年。隔离协议是基于在小胶质细胞和白细胞密度离心分离4。在这里，我们描述一个孤立的中枢神经系统的白细胞使用不连续percoll梯度的快速和有效的方法。经过细胞的分离，染色等各种抗体AS -的，但不局限于- CD45，CD4，CD8细胞CD11b，CD19等可以渗透后一个特定的病理诱导中枢神经系统的免疫人群的...

组织的同质化和渐变<ul><li> Percoll（Amersham公司） </li><li> 10X汉克斯“无钙，镁，平衡盐溶液（Gibco公司） </li><li>无酚红，中等的RPMI 1640（Gibco公司） </li><li> 7毫升或15毫升Dounce组织研磨机（VWR） </li></ul>流式细胞仪<ul><li>细胞染色缓冲液（Biolegend） </li><li>纯净的大鼠抗小鼠CD16/CD32（鼠标BD FC块），BD Pharmingen公司</li><li> Hemacytometer（费舍尔） </li><li>抗鼠CD45（克隆30 - F11），CD4（上克隆令吉- 5），CD19（克隆6D5），BioLegend </li><li> 10X磷酸盐缓冲液，无钙和镁（Thermo Scientific的） </li><li>多聚甲醛（Sigma公司Aldrich公司） </li><li>流式细胞仪分析与LSR II（BD公司） </li></ul>

isolation of brain and spinal cord mononuclear cells using percoll gradients

渗透在中枢神经系统感染，外伤，自身免疫性疾病或神经退行性疾病（CNS）的免疫细胞的分离，往往需要定义其表型和效应功能。组织化学方法有助于确定的浸润细胞的位置，并分析相关的中枢神经系统病理。然而，在原位组织化学及免疫荧光染色技术可以在一个单一的时间用来描述在一个特定的组织的免疫细胞亚型的抗体数量是有限的。因此，组织学与免疫表型流式细胞仪结合的方法是至关重要的，充分体现当地中枢神经系统浸润的组成。该协议是基于对中枢神经系统的细胞悬浮液比不连续percoll梯度分离。本文介绍一个快速的协议，有效地从大脑和脊髓组织，可以有效地利用各种免疫细胞群，在流式细胞仪检测单个样品的鉴定中分离出的单个核细胞。

Watch this Scientific Journal Video about Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients at JoVE.com

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

本文介绍一个快速的协议，有效地从大脑和脊髓组织，可以有效地利用流式细胞仪分析中分离出的单个核细胞。

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

单核细胞分离研究人类粘膜淋巴组织中先天免疫反应

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

使用微泡从人外周血单核细胞样品中分离、活化和扩增基于浮选的 T 细胞

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

PBMC Collection and Cryostorage for Mitochondrial Bioenergetics Analysis

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

Preservation of Bioenergetic Parameters in Peripheral Blood Mononuclear Cells After Cryopreservation (Video) | JoVE

人外周血单核细胞的冷冻保存与生物能量评价

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...

Histological Staining of Spinal Cord for Assessing EAE Disease Severity

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Unveiling the Pathway Linking Obesity to Autoimmune Inflammation in Multiple Sclerosis (Video) | JoVE

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...