| Murine Monocyte and Macrophage Culture
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Author:
Date:
2021-03-20
[Abstract] Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).
Graphic abstract: ...
[摘要] [摘要]骨髓中的骨髓祖细胞产生单核细胞，巨噬细胞，粒细胞和大多数树突状细胞。即使这些先天免疫细胞是同一谱系的一部分，每种细胞类型在组织发育，宿主防御和适应性免疫的产生中也发挥着特定而关键的作用。过去已经开发出区分骨髓细胞和骨髓细胞的协议，以进行功能研究并加深我们对它们对哺乳动物生理学贡献的理解。在该协议中，我们描述了一种简单快速的方法，可从鼠骨髓中分离单核细胞，将其培养长达5天，最后，将它们分化为源自骨髓的巨噬细胞（图1）。
图形摘要：
图1.实验概述，描绘了鼠单核细胞和巨噬细胞培养的步骤
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| 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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| Production and Isolation of Magnetic Protein Crystals in HEK293T Cells
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Author:
Date:
2020-07-20
[Abstract] Advances in protein engineering have enabled the production of self-assembled protein crystals within living cells. Our recent publication demonstrates the production of ftn-PAK4, which is a ferritin-containing crystal that can mineralize iron and become magnetic when isolated. We have developed an optimized protocol for the production and isolation of PAK4-based crystals. The crystals are first grown in low-passage HEK293T cells, released using a lysis buffer containing NP-40 and DNase, and collected under careful centrifugation conditions. Our protocol maximizes the purity and yield of crystals and is quick and straightforward.
[摘要] [摘要] 蛋白质工程的进展已使活细胞内产生自组装的蛋白质晶体。我们的最新出版物证明了ftn-PAK4的生产，它是一种含铁蛋白的晶体，在分离时可以矿化铁并成为磁性。我们已经开发出用于生产和分离基于PAK4的晶体的优化协议。晶体首先在低传代的HEK293T细胞中生长，使用含有NP-40和DNase的裂解缓冲液释放，并在仔细的离心条件下收集。我们的协议可最大程度地提高晶体的纯度和收率，并且操作简便快捷。
[背景] 近来的作品曾报道的“生产和隔离在纤维素的” 晶体通过蛋白在活细胞中的异源表达。这些晶体具有多种应用，例如货物运输（Ijiri 等，2009）或X射线结构测定（Baska ran 和Ang ，2015）。晶体的性质而变化，但它们通常相对于相当大的是对细胞，范围从1-2 微米到数百微米的大小（圣豪等人，2015） 。在我们最近的工作中，我们修改了inka-PAK4晶体以创建ftn-PAK4，该ftn-PAK4是一种含铁蛋白的晶体，可以矿化足够的铁以吸引附近的永磁体（Li 等，2019）。
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