大脑和脊髓的单核细胞的分离，使用Percoll梯度
Summary
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。
Abstract
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。
Protocol
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?…
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
Tissue homogenization and Gradients
- Percoll (Amersham)
- 10X Hanks’ Balanced Salt Solution, without Calcium and Magnesium (Gibco)
- RPMI Medium 1640, without phenol red (Gibco)
- 7-ml or 15-ml Dounce Tissue Grinders (VWR)
Flow cytometry
- Cell Staining Buffer (Biolegend)
- Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), BD Pharmingen
- Hemacytometer (Fisher)
- Anti-mouse CD45 (clone 30-F11), CD4 (clone RM4-5), CD19 (clone 6D5), BioLegend
- 10X Phosphate Buffered Saline, without Calcium and Magnesium (Thermo Scientific)
- Paraformaldehyde (Sigma Aldrich)
- Flow cytometry analysis performed with a LSR II (BD Biosciences)
Referenzen
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Tags
IsolationBrainSpinal CordMononuclear CellsPercoll GradientsImmune CellsCentral Nervous SystemInfectionTraumaAutoimmunityNeurodegenerationPhenotypeEffector FunctionsHistochemical ApproachesInfiltrating CellsCNS PathologyIn-situ HistochemistryImmunofluorescent Staining TechniquesAntibodiesImmune Cell SubtypesHistological ApproachesFlow CytometryCompositionCellular Suspensions
Diesen Artikel zitieren
Pino, P. A., Cardona, A. E. Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients. J. Vis. Exp. (48), e2348, doi:10.3791/2348 (2011).