| Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels
|
|
Author:
Date:
2020-11-05
[Abstract] Giant unilamellar vesicles (GUVs) are a widely used model system for a range of applications including membrane biophysics, drug delivery, and the study of actin dynamics. While several protocols have been developed for their generation in recent years, the use of these techniques involving charged lipid types and buffers of physiological ionic strength has not been widely adopted. This protocol describes the generation of large numbers of free-floating GUVs, even for charged lipid types and buffers of higher ionic strength, using a simple approach involving soft polyacrylamide (PAA) gels. This method entails glass cover slip functionalization with (3-Aminopropyl)trimethoxysilane (APTES) and glutaraldehyde to allow for covalent bonding of PAA onto the glass surface. After polymerization ...
[摘要] [摘要]巨型单层囊泡（GUV）是一种广泛使用的模型系统，其应用范围包括膜生物物理学，药物递送以及肌动蛋白动力学研究。虽然一些协议已经为他们这一代人在最近几年已开发，利用这些T的echniques涉及带电脂质的类型和生理离子强度缓冲液一直没有得到广泛的广告Ø PTED。Thi的方案描述了使用包括聚丙烯酰胺（PAA）凝胶的简单方法，即使对于带电荷的脂质类型和更高离子强度的缓冲液，也产生了大量的自由浮动GUV。此方法需要使用（3-氨基丙基）三甲氧基硅烷（APTES）和戊二醛对玻璃盖玻片进行功能化，以允许将PAA共价键合到玻璃表面上。PAA聚合后，将凝胶真空干燥。随后，将选择的脂质均匀地分散在干燥的凝胶表面上，并且可以使用具有不同离子强度的缓冲液来重新水化凝胶并形成GUV。该协议对于在生理条件下生产大量由不同脂质组成的自由浮动GUV而言是可靠的。它可以方便地用常用的实验室试剂进行。
[背景】虽然温和的水化和电铸是两个巨的最常用的方法单层囊泡（GUV）的形成，只有少数研究，报告其使用带电脂质类型和斯坦因的生理离子强度缓冲液（等人。，2017; ...
|
|
|
| Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells
|
|
Author:
Date:
2020-07-20
[Abstract] The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and ...
[摘要] [摘要] 骨骼肌是身体活动和运动表现的关键，但是衰老和疾病通常会由于肌肉细胞的浪费或变性而导致肌肉质量的逐步丧失。肌卫星细胞（MuSCs）代表的组织STE群体米细胞小号居住在骨骼肌和负责骨骼肌的体内平衡维持和再生。生长，损伤和变性信号激活MuSC，然后增殖（增殖的MuSC被称为成肌细胞），分化并与现有的多核肌肉细胞（肌纤维）融合，以介导肌肉的生长和修复。在这里，我们描述了从小鼠骨骼肌中分离MuSC的体外实验方案分析。此外，我们提供了有关如何将原代成肌细胞培养和分化成肌管的详细协议，以及用于表征细胞的免疫荧光染色程序。这些方法对于在体外模拟再生肌生成以了解MuSC 的动力学，功能和分子调控至关重要。
[背景] 通过多种细胞功能维持肌肉的动态平衡，对于保持肌肉的完整性至关重要。组织特异性成体干细胞能够在整个生命中连续不断地再生局部组织。在成年骨骼肌中，称为肌肉卫星细胞（MuSC）的干细胞群具有强大的再生能力，这是肌肉动态平衡的关键（Yin 等人，2013； Dumont 等人，2016）。静态MuSC位于与肌肉纤维并列的基底层下方的壁iche中，负责肌肉的生长和再生（Yin 等人，2013； Dumont 等人，2016）。
...
|
|
|