| Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System
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Author:
Date:
2020-03-05
[Abstract] T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a ...
[摘要] [摘要] T细胞是免疫系统的一种主要细胞类型，利用其T细胞抗原受体（TCR）结合并响应源自病原体的外来分子。配体-TCR相互作用的半衰期决定了刺激的结果。直到最近，科学家还是依靠突变TCR或其配体来研究变化的TCR-配体相互作用持续时间如何影响T细胞活化。我们新创建的光配体-TCR系统使我们能够通过光照精确且可逆地控制配体与TCR的结合。该系统使用植物色素B（PhyB ）四聚体作为光调节的TCR配体。PHYB 可光转化的结合（ON）之间，并且通过分别为660nm和740nm的光照射，非结合性（OFF）构象。PhyB ON能够结合通过将PhyB 相互作用因子（PIF）融合到TCRβ链而产生的合成TCR 。将PhyB 切换为OFF构象会破坏这种相互作用。PhyB 四聚体与PIF-TCR 足够长的结合导致T细胞活化（通过钙内流测量）。在这里，我们描述了有关如何为我们的光配体-TCR系统生成四聚体配体，如何通过流式细胞仪测量配体-TCR结合以及如何通过钙内流量化T细胞活化的协议。
[背景 ] ...
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| Isolation, Culture, and Staining of Single Myofibers
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Author:
Date:
2016-10-05
[Abstract] Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single ...
[摘要] 成年骨骼肌再生由被称为卫星细胞的成人干细胞的专门群体协调，其被定位在基底层和肌纤维的质膜之间。在肌肉再生期间发生的卫星细胞激活，增殖和随后分化的过程可以通过从骨骼肌中分离单个肌纤维并在悬浮条件下培养它们而在体外重现。在这里，我们描述了一种改进的协议，通过从小鼠的伸肌腱长肌（EDL）肌肉中分离单个肌纤维来评价离体细胞的活化，并培养和染色肌纤维相关的卫星细胞的标记物自我更新，增殖和分化。
[背景] 虽然骨骼肌是一种完全分化的有丝分裂后组织，但是它保持了内在的能力，以对遗传和获得性肌肉纤维损伤的形式Grand和Rudnicki，2007）。成年人的肌肉再生由称为卫星细胞的干细胞群体介导，所述细胞位于有丝分裂静止状态的肌纤维的基底层和肌纤维膜之间（Le Grand和Rudnicki，2007）。响应于肌肉创伤，卫星细胞变得活化和增殖以产生与预先存在的纤维融合的成肌细胞，并彼此相互修复或产生新的肌纤维。一小部分卫星细胞不分化，而是重新进入静止以维持干细胞库。所有哺乳动物物种的卫星细胞表达配对盒（Pax）转录因子Pax7，其也用作确定与其他生肌因子如MyoD相关的卫星细胞命运的关键标记（Le Grand和Rudnicki，2007; et al。，2006; Kuang and Rudnicki，2008）。
可以通过肌纤维外植体的悬浮培养部分地重现关于卫星细胞活化，增殖和分化的肌肉再生的体内过程（Rosenblatt等人，1995; ...
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| Calcium Mobilisation Assay in Response to Chemokine Stimulation
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Author:
Date:
2012-05-05
[Abstract] This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, catalog number: 211842) in the loading buffer (at 100 μM) allows for quantification using the FLIPR system on poly-D-lysine plates. Probenicid is an anion-exchange protein inhibitor and prevents the extrusion of the dyes by organic ion transporters.
[摘要] 这篇文章所述分析是应用参比分析来测量趋化因子刺激下淋巴细胞（原代细胞或细胞株）中钙离子含量的变化。它也被用于测量T细胞受体介导的钙通量。另外，带有外加溶于上样缓冲液（浓度100 μM）的亮黑（一种淬灭剂，Sigma公司，编号 # 211842）的相同标签程序允许在多聚D-赖氨酸板上定量使用FLIPR 系统。羧苯磺胺是一种阴离子交换蛋白抑制剂，能防止有机离子转运蛋白挤压燃料。
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