| Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice
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Author:
Date:
2017-05-05
[Abstract] Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been ...
[摘要] 造血是一种多步骤过程，导致在损伤的肌纤维的胚胎发育和修复期间骨骼肌的形成。在这个过程中，成肌细胞是主要的效应细胞类型，彼此融合或损伤肌纤维，导致新成肌纤维的形成或成年人骨骼肌的再生。通过体外成骨细胞分化成肌管可以概括出许多发生肌肉发育的步骤。大多数实验室使用也分化成肌管的永生化肌原细胞系。虽然已经发现这些细胞系对于描绘造血的调节机制非常有用，但是它们通常依赖于细胞的来源和培养条件而显示出很大的变异性。原代成肌细胞被认为是体外研究肌生成的最生理学相关模型。然而，由于成体骨骼肌的丰度低，原代成肌细胞的分离在技术上是有挑战性的。在本文中，我们描述了一种用于从小鼠的成年骨骼肌分离原代成肌细胞的改进方案。我们还描述了其培养和分化成肌管的方法。
背景 造血是一个复杂而高度协调的过程，其涉及多潜能中胚层细胞的测定，以产生成肌细胞，成肌细胞从细胞周期中排出，以及它们最终分化为骨骼肌纤维。 Myogen-5，MyoD，myogenin和MRF4的基因螺旋 - 环 - 螺旋转录因子的一组基因调控因子（MRFs）的顺序表达调控。 Myf-5和MyoD是成肌细胞形成，增殖和存活所需的主要MRFs，而其他MRF（如肌细胞生成素和MRF-4）在肌发生过程中起作用迟发，激活收缩蛋白和其他结构和代谢蛋白的基因表达（白金汉，2003; ...
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| FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
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Author:
Date:
2015-08-20
[Abstract] Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal ...
[摘要] 荧光活化细胞分选（FACS）是用于从成年小鼠骨骼肌纯化卫星细胞或肌肉干细胞的灵敏和精确的方法（Liu等人，2013; Sacco等人， ，2008; Tierney ，，2014）。 后肢肌肉的机械和酶消化将单核肌细胞释放到悬浮液中。 该方案采用分级分离策略，通过磁性分离和基于FACS的排除，耗尽表达细胞表面标记CD45，CD31，CD11b和Ly-6A/E-Sca1的细胞，并阳性选择表达α7-整联蛋白和CD34的细胞。 这使得研究者能够成功地富集均匀表达配对盒转录因子Pax7并且能够长期自我更新，骨骼肌修复和肌肉干细胞池再增殖的卫星细胞。
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| Extravillous Trophoblast Migration and Invasion Assay
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Author:
Date:
2013-08-05
[Abstract] Extravillous trophoblast (EVT) migration and invasion through the decidualized endometrium is essential to successful placentation. SGHPL-4 cells, an EVT cell line derived from first trimester placenta, is a widely used model of cytotrophoblast differentiation into an invasive phenotype. Here we describe a quantitative cell migration assay that can be modified to also measure cell invasion. SGHPL-4 cells were seeded into BD Fluoroblok cell culture inserts constructed with an 8 μm porous membrane and allowed to migrate towards epidermal growth factor, a known chemoattractant for EVTs. To assess EVT invasion, Fluoroblok inserts were first coated with Matrigel, a basement membrane matrix. SGHPL-4 cells were labeled with calcein AM and cells that had invaded and/or migrated across the ...
[摘要] Extravillous滋养层（EVT）迁移和通过蜕膜化子宫内膜的侵入是成功胎盘成形的关键。 SGHPL-4细胞，来自前三个月胎盘的EVT细胞系，是广泛使用的细胞滋养层分化为侵袭性表型的模型。在这里我们描述了一种定量细胞迁移实验，可以修改也测量细胞入侵。将SGHPL-4细胞接种到用8μm多孔膜构建的BD Fluoroblok细胞培养插入物中，并允许其向表皮生长因子（一种已知的EVTs化学吸引剂）迁移。为了评估EVT侵入，首先用Matrigel（基底膜基质）包被Fluoroblok插入物。用钙黄绿素AM标记SGHPL-4细胞，并通过底部读数荧光读板器定量已经侵入和/或迁移穿过膜的细胞。 Fluoroblok插入物优于其他迁移/侵袭测定的优点是它们允许迁移细胞的非破坏性检测。
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