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HyClone Penicillin-Streptomycin 100X solution

青霉素 - 链霉素100X溶液

Company: GE Healthcare
Catalog#: SV30010
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Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
Author:
Date:
2017-09-05
[Abstract]  The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system. The P2X7 receptor is expressed in Sf9 insect cells as a GFP fusion protein and solubilized with a buffer containing Triton X-100 detergent. The P2X7-GFP fusion protein is ... [摘要]  P2X7受体是仅在真核生物中发现的胞外ATP门控离子通道(Bartlett等,2014)。由于其P2X受体之间的独特性质,例如大电导孔的形成,P2X7受体已经涉及破坏性疾病如慢性疼痛(North和Jarvis,2013)。然而,P2X7特异性属性的机制仍然知之甚少,部分原因是纯化这种真核膜蛋白是一个挑战。在这里,我们描述了使用昆虫细胞 - 杆状病毒系统表达和纯化哺乳动物P2X7受体的详细方案。 P2X7受体在作为GFP融合蛋白的Sf9昆虫细胞中表达,并用含有Triton X-100洗涤剂的缓冲液溶解。然后使用Strep-Tactin亲和层析在含有十二烷基麦芽糖苷的缓冲液中纯化P2X7-GFP融合蛋白。在通过凝血酶酶切割连接的GFP和Strep-标签后,使用大小排阻色谱分离P2X7受体。该方法通常从6L的Sf9培养物产生约2mg的纯化蛋白质。纯化的蛋白质可以用含有15%甘油的缓冲液在4℃下储存至少2个月,并用于各种功能和结构研究(Karasawa和Kawate,2016)。
【背景】P2X7受体是嘌呤能P2X受体家族的七种亚型之一,并且是广泛疾病如神经退行性疾病,癫痫和神经性疼痛的有希望的新型药物靶点(North和Jarvis,2013; Bhattacharya和Biber, ...

Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells
Author:
Date:
2017-07-05
[Abstract]  Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to distant cells, thereby affecting their gene expression, proliferation, differentiation, and function. Although their physiological functions are not clearly defined, recent studies have shown their therapeutic potential for tissue repair and regeneration. While MVs can be isolated readily from mesenchymal stem cells (MSCs) and other cell types from various sources, the yield of MVs under conventional culture condition in vitro is one of the limiting factors for both the in ... [摘要]  微囊泡(MV)是亚微米尺寸的膜泡囊,其通过分泌室从细胞中积极释放或从细胞表面膜脱落。 MV由许多细胞类型产生并且用作将生物信息(例如,蛋白质,mRNA和miRNA)转移到远端细胞的载体,从而影响其基因表达,增殖,分化和功能。 虽然他们的生理功能没有明确定义,但最近的研究已经显示出其组织修复和再生的治疗潜力。 虽然MV可以从间充质干细胞(MSC)和来自各种来源的其他细胞类型容易地分离,但在体外常规培养条件下MV的产量是限制因素之一, 功能研究以及体外分析分析。 在这里,我们提供了一个通过大鼠脑提取物预处理MSC增加微泡产量的方案。
【背景】通过直接重编程或利用间充质干细胞进行细胞替代治疗来产生神经干细胞或神经细胞是神经变性疾病的潜在选择(Adib等人,2015)。最近的研究已经证明,来自MSC的微泡代表了增强组织再生,例如神经元再生,免疫调节,脑损伤中的血管发生的其他细胞替代方法的新颖且安全的替代方案(Kim等人,2013) ; Porro等,,2015; Lee等人,2016)。对受损组织外源信号如何影响微泡数量和组成的了解甚少。 MSCs的功能分泌物的含量和数量可以根据微环境的显着变化(Qu等人,2007)。例如,已知缺血性脑提取物或缺氧诱导合成有益于组织再生过程的许多细胞因子和生长因子(Chen等,2007; Shin et ...

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells
Author:
Date:
2017-04-20
[Abstract]  Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters.

This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following ...
[摘要]  细胞条形码可以通过单细胞谱系追踪来分离异种细胞群体中的克隆动力学。通过独特和可遗传的DNA条形码对造血干细胞和祖细胞(HSPC)进行标记,可以通过随后的移植和高通量测序,在单细胞水平上分化供体细胞异质性,分化潜力和谱系偏倚。此外,细胞条形码可以根据功能而不是免疫表型参数定义真正的造血干细胞(HSC)。
 该协议描述了慢病毒细胞条形码的工作流程,追踪1450天后(dpc)胎肝(FL)Lineage-Sca+ cKit + (LSK)HSPC经过长期重建(图1)(Kristiansen等人,2016),但可以适应于选择的细胞类型或时间框架。


图1.实验工作流程摘要(Naik 等人,2013)

最初建立了细胞条形码技术来解决在体内移植造血细胞后的单细胞动力学,并且近年来在移植中对血细胞群体中功能​​异质性的认识有显着贡献(Schepers ,2008; Gerrits等人,2010; Lu等人,2011; Naik等人 ,2013; Verovskaya等人,2013; ...

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