A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms
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Author:
Date:
2020-09-20
[Abstract] This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher ...
[摘要] [摘要] 该协议描述了一种简单的方法,可在滤纸中冷冻保存哺乳动物细胞,以替代常规的慢速冷冻方法。该方法包括使用纤连蛋白处理纸纤维,使用低浓度的冷冻保护剂二甲基亚砜(DMSO),然后以1°C min -1的速率将细胞缓慢冷冻至-80°C 。在我们的方法中,纸的生物相容性,大表面积,3D孔隙率和纤维柔韧性与纤连蛋白处理相结合,可产生与传统方法相当的细胞回收率,而无需对冷冻和解冻程序进行额外的微调。我们期望纸质冷冻保存方法这将为保存哺乳动物细胞领域带来几项优势,包括在单位体积内容纳更多数量的细胞,并且释放后无细胞损失。该方法需要最小的存储空间,在该存储空间中,可以将具有大面积的纸平台卷起和/或折叠并存储在库存中,并允许按需方式有效地运输/分配细胞。此外,该方法的另一个特征包括细胞球体和3D细胞培养物的形成和冷冻保存。
[背景] 哺乳动物细胞的成功保存,长期保存,维护和分配是重要的研究领域,目前仍在深入的科学研究中。特别是,冷冻细胞的及时稳定供应与组织工程研究有关,例如细胞培养,药物开发和测试以及再生和生物治疗医学。
当前的常规细胞冷冻保存方案包括缓慢和快速的冷冻和玻璃化(Pegg,2002; Baust 等,2009)。在这些方法中,将各种浓度的冷冻保护剂添加到细胞悬浮液中,然后以低至1°C min -1 ...
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Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy
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Author:
Date:
2018-04-05
[Abstract] One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into Xenopus laevis oocytes is ideally suited for this purpose. Xenopus laevis oocytes are a commonly used eukaryotic cell model in different fields of biology, such as cell- and development-biology. For in-cell EPR, the bio-macromolecules of interest are microinjected into the Xenopus laevis oocytes upon site-directed spin labeling. The sample solution is filled into a thin glass capillary by means of Nanoliter Injector and after that ...
[摘要] 研究生物大分子的最令人兴奋的观点之一来自于新兴的细胞内光谱学领域,它能够确定细胞中生物大分子的结构和动力学。细胞内电子顺磁共振(EPR)光谱结合将生物大分子微注射到非洲爪蟾卵母细胞中非常适合于此目的。非洲爪蟾卵母细胞是生物学不同领域常用的真核细胞模型,如细胞和发育生物学。对于细胞内EPR,感兴趣的生物大分子通过定点自旋标记显微注射到非洲爪蟾卵母细胞中。通过Nanoliter注射器将样品溶液填充到薄玻璃毛细管中,然后通过小心地穿刺薄膜将其微注射入非洲爪蟾卵母细胞的黑色动物部分。之后,取决于最终的细胞内EPR实验的种类,将三个或五个显微注射的非洲爪蟾卵母细胞装载到Q波段EPR样品管中,随后进行任选的休克冷冻(用于实验冷冻溶液)并且在期望的温育时间之后测量(在低温或生理温度下)。由于显微注射样品的细胞毒性作用和顺磁性自旋标记在还原性细胞环境中的稳定性,孵育时间受到限制。通过监测细胞形态和减少动力学来量化这两个方面。
【背景】电子顺磁共振(EPR)光谱学是用于表征顺磁系统的选择方法(Atherton,1993; Gerson等人,1994; Jeschke和Schweiger,2001)。反磁性生物大分子可以通过定点自旋标记(SDSL)进行EPR光谱学分析,通常使用氮氧化物作为自旋标记(Hubbell和Altenbach,1994; Feix和Klug,2002; ...
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Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines
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Author:
Date:
2018-03-20
[Abstract] The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.
[摘要] 主要纤毛是一种非运动感觉细胞器,其装配和拆卸与细胞周期进程密切相关。 初级纤毛在静止细胞中从基体拉长并随着细胞重新进入细胞周期而被吸收。 睫状动力失调与纤毛病和其他人类疾病有关。 体外血清刺激的睫状体装配/分解测定已经在解决睫状动力学中感兴趣的蛋白质的功能方面受到欢迎。 在这里,我们描述了转染人视网膜色素上皮细胞(RPE-1)和对转染细胞进行睫状体装配/分解测定的充分测试的方案。
【背景】初级纤毛是毛发样感觉细胞器,其在G 0 / G 1期出现,并且在细胞周期的S期之前分解(Tucker等, et al。,1979)。先前的研究已经证实,某些未转化的细胞类型(即,甚至是RPE-1细胞,3T3成纤维细胞和小鼠胚胎成纤维细胞[MEFs])可以被饿死以诱导静止和睫状体形成。随后的血清再次添加触发双相睫状体吸收,其在刺激后2小时和24小时达到峰值(Tucker等人,1979; Li等人,2011) 。该现象为文献中常用的血清刺激的睫状体组装/分解测定奠定了基础,以鉴定参与睫状体组装和拆卸的蛋白质(Pugacheva等人,2007; ...
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