Name: expression of fluorescent fusion proteins in murine bone marrow-derived dendritic cells and macrophages
Uploaded: 2018-10-30T12:30:00
Description: Watch this Scientific Journal Video about Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages at JoVE.com

这项工作得到了捷克科学基金会 (GACR 项目编号 16-07425S) 的支持, 由查尔斯大学赠款机构 (GAUK) (项目编号 923116) 和捷克科学院分子遗传学研究所的机构资助。共和国 (RVO 68378050)。

这里描述的所有方法都是由分子遗传学研究所的实验动物福利问题专家委员会和捷克共和国科学院批准的。
1. 试剂制备
<ol><li>制备氯化铵-钾 (ACK) 缓冲液。添加 4.145 g 的 NH4Cl 和 0.5 g 的 KHCO3到500毫升的 ddH2O, 然后添加100µL 的 0.5 M 乙酸酸 (EDTA) 和过滤消毒。</li>
	<li>准备聚乙烯亚胺 (PEI) 解决方案。加入0.1 克 PEI 到90毫升的 ddH2O。搅拌时, 在 pH 值?...

<ol>
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目标细胞中蛋白质的表达是许多生物研究的关键步骤。通过标准转染和逆转录病毒转导技术很难转染分化的巨噬细胞和树突状单元。绕过这些分化细胞的转染骨髓祖的逆转录病毒转导, 其次是分化时, 他们已经携带所需的结构, 是一个关键步骤, 允许在这些 cDNAs 的表达单元格类型。成功使用此方法的一个例子可以在我们最近的出版物25中找到。在这里, 我们提供了一个经济高效的协...

髓细胞是我们抵御病原体的防御机制不可或缺的一部分。他们能够迅速消除微生物, 以及垂死的细胞。此外, 他们还参与调节组织的发展和修复, 并保持稳态1,2,3。所有髓细胞均与骨髓中的常见髓内祖先分化。它们在许多功能和形态上不同的子集的分化是在很大程度上由细胞因子和它们的各种组合控制4。最深入研究的髓细胞亚群包括中性粒细胞粒细胞、巨细胞和树突状体细胞。任何这些人群中的缺陷会导致潜在的危及生命的后果, 并导致人类和小鼠免疫系统的严重功能失调1,2,3,5,6。
与中性粒细胞粒细胞不同的是, 树突状体细胞和巨噬细胞是组织的驻留单元, 免疫器官的丰度相对较低。因此, 在需要大量这些细胞的实验中, 分离和纯化原代树突状细胞和巨细胞是昂贵的, 而且往往是不可能的。为了解决这一问题, 已经开发出了一种在体外获得大量同质巨噬细胞或树突胞的实验方案。这些方法的基础是在细胞因子存在下的小鼠骨髓细胞分化: 巨噬细胞集落刺激因子 (M CSF) 用于巨噬细胞和粒细胞-巨噬 Flt3 集落刺激因子 (GM-csf) 或树突状细胞7,8,9,10,11,12。这种方法产生的细胞通常在文献中描述为骨髓衍生巨噬细胞 (BMDMs) 和骨髓衍生树突状细胞 (BMDCs)。它们与原生巨噬细胞或树突状干细胞的共同生理特性高于相应的细胞系。另一个主要优势是获得这些细胞形成转基因小鼠13的可能性。从转基因小鼠中提取的野生型细胞和细胞之间的比较研究对于揭示基因或感兴趣的蛋白质的新功能通常是至关重要的。
在活细胞中蛋白质的亚细胞定位分析需要荧光标签与体内感兴趣的蛋白质的耦合。这通常是通过表达由分析的蛋白质耦合 (通常通过短链接器) 与荧光蛋白 (例如, 绿色荧光蛋白 (GFP))14、15的基因编码的融合结构来实现的。,16. 荧光标记蛋白在树突状细胞或巨噬细胞中的表达具有挑战性。这些细胞通常难以转染的标准转染程序和效率往往是非常低的。此外, 转染是瞬态的, 它产生细胞应力和达到的荧光强度可能不足以显微镜17。为了获得足够的转基因表达水平的这些细胞的合理分数, 骨髓祖细胞与逆转录病毒载体的感染及其随后的分化成 BMDMs 或 BMDCs 已成为一个非常有效的方法。它允许对其原生细胞环境中的髓样蛋白进行分析, 无论是在稳态还是在对免疫反应至关重要的过程中, 如吞噬、免疫突触形成或迁移。在这里, 我们描述一个协议, 允许在小鼠骨髓巨噬细胞和树突状细胞中稳定表达荧光标记蛋白的兴趣。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>15028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>10270</td>
			<td>For media suplementation</td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Lachema, Brno, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>BB Pharma AS, Prague, Czech Republic</td>
			<td>N/A</td>
			<td>PENICILIN G 1,0 DRASELN&#193; SOL&#39; BIOTIKA</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>S9137</td>
			<td>Streptomycin sulfate salt powder</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Dr. Kulich Pharma, Hradec Kr&#225;lov&#233;, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, linear, MW 25,000</td>
			<td>Polyscience, Warrington, PA, USA</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Prepared in-house by media facility of IMG ASCR, Prague, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse/human CD11b Antibody, clone M1/70</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>101212</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80 Antibody, clone BM8</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>123110</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD11c Antibody, clone N418</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>117310</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech (Rocky Hill, NJ, USA)</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>PeproTech (Rocky Hill, NJ, USA)</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>H1398</td>
			<td>flow cytometry analysis use at 1-2 &#181;g/ml</td>
		</tr>
	</tbody>
</table>

expression of fluorescent fusion proteins in murine bone marrow-derived dendritic cells and macrophages

树突状细胞和巨噬细胞是形成防御病原体的第一道防线的关键单元。它们也在启动自适应免疫反应中发挥重要作用。与这些细胞的实验工作是相当困难的。它们在器官和组织中的丰度相对较低。因此, 它们不能被大量隔离。它们也很难与 cDNA 构造转染。在小鼠模型中, 这些问题可以部分克服由骨髓祖细胞存在的细胞或 GM 脑脊液的树突状干细胞的体外分化。这样, 就可以从很少的动物那里获得大量这些细胞。此外, 骨髓祖细胞可以转导与逆转录病毒载体, 在其分化为骨髓衍生树突状干细胞和巨噬细胞之前, 在早期培养阶段进行 cDNA 构造。因此, 逆转录病毒转导后的体外分化可用于表达这些细胞中的各种 cDNA 结构。表达异位蛋白的能力大大扩展了可在这些细胞上进行的实验范围, 包括荧光蛋白的活细胞成像、路特分析的串联纯化、结构-功能分析、利用生物传感器和其他许多功能监测蜂窝细胞的机能。在本文中, 我们描述了一个详细的协议的逆转录病毒转导的小鼠骨髓衍生树突状细胞和巨噬细胞与载体编码的荧光标记蛋白。以两个适配器蛋白、OPAL1 和 PSTPIP2 为例, 说明其在流式细胞仪和显微术中的实际应用。我们还讨论了这种方法的优点和局限性。

信号适配器蛋白通常是小蛋白质, 没有任何酶活性。它们具有各种相互作用的领域或图案, 它调解与信号传导相关的其他蛋白质的结合, 包括酪氨酸激酶、磷酸酶、泛素连接酶等21。为演示此协议的功能骨髓细胞适配器 PSTPIP2 和 OPAL1 被选择。PSTPIP2 是一个很好的特征蛋白参与炎症反应的调节22。它是一种细胞质蛋白, 也可以通过其 F bar 域被...

Watch this Scientific Journal Video about Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages at JoVE.com

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

本文详细介绍了荧光融合蛋白在小鼠骨髓树突状细胞和巨噬细胞中的表达。该方法以骨髓祖细胞转导为基础, 采用逆转录病毒构造, 然后在体外分化成巨噬和树突状体细胞。

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

该方法有助于回答巨噬细胞和树突细胞生物学领域有关这些细胞类型中各种蛋白质的亚细胞定位和动力学的关键问题。这种技术的主要优点是，它相对简单，价格低廉，并且比大多数其他可用方法表现出更好的表达效率和稳定性。演示这个程序的将是贾米拉·克拉洛娃，一个研究生从我的实验室。为了产生逆转录病毒，在10厘米的培养皿中将铂金生态包装细胞的单细胞悬浮板，并在15毫升DMEM中培养细胞，辅以10%的胎儿牛血清或FBS，直到培养量为50%至60%的汇合。第二天，添加20微克的兴趣的逆转录病毒构造和10微克的PCL生态包装载体到1毫升DMEM没有血清，与温和的混合。在1毫升DMEM中加入75微升的PEI，无需血清和抗生素，在第二管中，在室温下孵育5分钟，并将两个管的内容混合在一起，在室温下再孵育10分钟。接下来，小心地用8毫升的新鲜37摄氏度DMEM和2%FBS补充的铂金生态细胞上的介质，小心地将转染混合物加入到板材中。在37摄氏度下孵育4小时后，用10毫升37摄氏度DMEM代替超级支架，辅以10%FBS，在37摄氏度下24小时孵育。第二天，使用5毫升移液器将含有生态抗逆转录病毒颗粒的超高支架转移到15毫升离心管中，通过离心去除碎屑和含有细胞。同时，在培养皿中加入10毫升37摄氏度DMEM加10%FBS，在37摄氏度下再进行24小时孵育，并收集第二轮病毒颗粒，正如刚刚证明的。要收获骨髓，在组织培养罩中，使用钳子和剪刀将皮肤和部分肌肉从后肢中去除，并小心地将乙酰氨从臀部关节上除去，而不会打碎股骨。切开脚踝关节的爪子，用70%乙醇喷洒骨头。然后用纸巾取出肌肉的其余部分，将骨骼放在 PBS 的 5 厘米培养皿中，并辅以 2%FBS。接下来，弯曲每个骨骼的膝关节，用剪刀小心地分离骨骼。使用剪刀去除一根骨头的大约一到两毫米，并使用两到五毫升注射器，内装有30个量表针，并装有新鲜的PBS和FBS，将骨髓从骨的两端冲洗成15毫升的离心管。从每个骨骼中采集骨髓后，通过离心对骨细胞进行颗粒状，并在2.5毫升红血球解液中重新悬浮该颗粒，在室温下两到三分钟。在开水后，通过100微米细胞过滤器将细胞过滤到一个新的15毫升离心管中，用12毫升PBS加FBS恢复坚韧。在DMEM中重新悬浮颗粒，辅以10%FBS和抗生素计数，并板5至10倍10至第六骨髓细胞在10厘米，非组织培养处理培养培养的培养培养在10毫升DMEM的培养，辅以血清和巨噬菌体组刺激因子，或MCSF。在培养的第五天，小心地倾斜细胞培养皿，从靠近培养皿边缘的表面取出除约约5毫升的介质外的所有培养剂。然后加入20毫升的新鲜37摄氏度的培养，辅以FBS和MCSF到培养皿中，并将细胞返回细胞培养箱。为了收获细胞，第6至第7天，取出所有中和浮动细胞，用37摄氏度的PBS清洗培养物。在组织培养培养箱中，用5毫升0.02%EDTA和PBS分离粘附细胞，在37摄氏度下3至5分钟，并使用5毫升移液器轻轻地将分离的细胞从板底冲洗。然后将细胞转移到一个50毫升的离心管，其中包含25毫升的新鲜PBS。为了诱导EGFP标记蛋白表达在骨髓衍生抗原呈现细胞，在细胞分化方案开始时，稀释骨髓细胞到2至5倍10至6细胞每毫升DMEM加FBS和抗生素结合和板细胞的一毫升中等每井，辅以适当的分化细胞因子。在细胞培养孵化器中四到六个小时后，加入第一轮收集的病毒的两毫升，辅以多布雷尼，通过离心和细胞培养孵化器四小时的孵化来转导细胞培养。在转导期结束时，将非稳定细胞从每个井转移到单独的15毫升离心管中，通过离心收集细胞。如果需要大量细胞，细胞可以在多个孔中感染相同的收缩，并在分化前进行汇集。然后重新悬浮在培养基的10毫升颗粒，辅以适当的分化细胞因子，并板将细胞到每管一个10厘米的非组织培养皿上，为它们6至8天的培养，正如刚刚证明的。通过流量细胞学对铂金生态转染的疗效进行评估，在收集了含有超毒的二号病毒后，发现PSTPIP2-EGFP的均转染效率为62%，OPAL1-EGFP的转染效率为53%。逆病毒转导不影响骨髓衍生细胞的分化状态，两种培养物中超过90%的细胞在转导后对各自的标记物表现出积极。并且通过两种分化细胞的合适、有特征的形态变化观察。值得注意的是，与骨髓衍生的巨噬细胞EGFP表达相比，EGFP在骨髓衍生树突状细胞培养中的整体表达较低，而无论逆转录病毒的类型如何。在尝试此过程时，重要的是要记住使用非组织、培养处理的塑料盘，如果您计划从培养皿中去除细胞进行实验，通常用于培养细菌。按照这个程序，其他方法活细胞成像，超分辨率显微镜或其他类型的显微镜可用于回答有关树突细胞和感兴趣的巨噬细胞蛋白质的亚细胞定位和动力学的其他问题。这项技术为免疫学领域的研究人员探索巨噬细胞和树突状细胞表达的蛋白质的分布和空间关系，以及加深我们对这些蛋白质在免疫反应过程中的功能的理解铺平了道路。不要忘记，使用病毒颗粒可能极其危险，在执行此过程时，应始终采取预防措施，例如将气溶胶生成降至最低、佩戴防护设备以及使用层流罩。

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

在活细胞荧光蛋白的双探头光学突出纯净的光转化的

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...