| A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells
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Author:
Date:
2017-08-20
[Abstract] The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability of MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for ...
[摘要] 认为基于间充质干细胞（MSC）的细胞疗法的有益作用主要是由能够抑制慢性或急性损伤，感染，自身免疫和移植物抗宿主病相关炎症的能力介导的。 为了专门解决由血流或壁剪应力（WSS）引起的摩擦力对人MSC免疫调节功能的影响，我们利用微流体在动脉腔壁上建模WSS。 随后通过在共培养测定中通过活化的小鼠脾细胞测量TNF-α产生来量化MSC的抗炎效力。 TNF-α抑制测定作为MSC效力的功能评估的可重现平台，并且表现出作为MSC治疗功效的替代测定的预测价值。
【背景】间充质干细胞（MSC）的免疫调节活性由直接细胞相互作用和旁分泌因子介导（Singer和Caplan，2011;英语，2013）。 MSCs被认为是源于与骨髓和各种组织内脉管系统内皮细胞相关的周细胞（Sacchetti et al。，2007; Crisan et al。，2008）。这种独特的血管周围位置将它们置于血流中的炎症和其他可溶性因子附近，使其监测系统信号。事实上，将壁壁细胞募集到内皮是血管成熟的关键事件，周细胞在血管维持和完整性中起关键作用（Benjamin et al。，1998; ...
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| Measuring Auxin Transport Capacity in Seedling Roots of Medicago truncatula
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Author:
Date:
2016-06-20
[Abstract] Measurement of auxin transport capacity provides quantitative data on the physiological machinery involved in auxin transport within plants. This technique is easy to perform and gives quick results. Radiolabelled auxin (indole-3-acetic-acid) is fed into the roots of Medicago truncatula via an agar block. The resulting radioactivity from radiolabelled auxin uptake in the roots is measured with a liquid scintillation counter. Here, we describe the measurement of auxin transport capacity around the nodulation susceptible zone in young seedling roots of M. truncatula in response to rhizobia inoculation. Similar assays could be adapted in other plant species and to answer other biological questions.
[摘要] 生长素运输能力的测量提供了涉及植物中生长素运输的生理机制的定量数据。 这种技术很容易执行，并给出快速的结果。 将放射性标记的生长素（吲哚-3-乙酸）通过琼脂块加入到Medic藜苜蓿的根部。 使用液体闪烁计数器测量根中放射性标记的生长素摄取的最终放射性。 在这里，我们描述了生长素运输能力周围的结瘤敏感区在年轻幼苗根的测量。 truncatula 响应根瘤菌接种。 类似的测定可以适用于其他植物物种并回答其他生物学问题。
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| Macrophage Inflammatory Assay
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Author:
Date:
2014-07-20
[Abstract] Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al. ...
[摘要] 巨噬细胞代表广泛分布的和功能不同的先天骨髓细胞群，参与对病原体的炎症反应，组织内稳态和组织修复（Murray和Wynn，2011）。巨噬细胞可以大致分为具有相反活性的两个亚群：M1或促炎性巨噬细胞，其促进T辅助1型（Th1）细胞免疫和组织损伤，以及M2或抗炎或交替激活的巨噬细胞涉及Th2反应和分辨率的炎症。在这里我们描述了一种快速测定，我们以前用于监测由脂多糖（LPS）激活的巨噬细胞在响应于由成体干细胞产生的治疗性旁分泌因子的促炎和抗炎细胞因子产生中的变化（Bartosh等，/em>，2010; Ylostalo等人，2012; Bartosh 等人，2013）。该测定可以适当地适应于测试巨噬细胞对其它试剂的响应，在本文中将称为"测试试剂"或"测试化合物"。在该方案中，小鼠巨噬细胞细胞系J774A.1被扩增作为陪替氏培养皿上的粘附单层，允许容易地收获细胞而没有可以损伤细胞的酶或细胞刮擦器。然后用LPS悬浮刺激大孔，并接种到含有试验试剂的12孔细胞培养板中。 16-18小时后，收集由巨噬细胞调节的培养基，并用酶联免疫吸附测定（ELISA）测定培养基中的细胞因子谱。我们常规测量促炎细胞因子TNF-α和抗炎细胞因子白细胞介素-10（IL-10）的水平。
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