Name: economical and efficient protocol for isolating and culturing bone marrow-derived dendritic cells from mice
Uploaded: 2022-07-01T12:30:00
Description: Watch this Scientific Journal Video about Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice at JoVE.com

本研究由天津市科技计划（20JCQNJC00550）、天津市卫生科技计划（TJWJ202021QN033和TJWJ202021QN034）资助。

所有程序均经南京医科大学动物保育使用委员会批准。
1. 骨髓分离和BM细胞的制备
<ol><li>通过CO 2窒息处死C57BL / 6小鼠（18-22g ，6-8周龄）。将鼠标固定在鼠标操作台上。用70%乙醇消毒表面。</li>
	<li>切开腿部皮肤以暴露肌肉和股动脉。用两个镊子夹住并撕下股动脉，然后将近端拉向腹部。 注意：不要直接切除股动脉。否则，会导致出血过多，污染视野?...

<ol>
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L., Maliszewski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+murine+dendritic+cells+from+flt3-ligand-supplemented+bone+marrow+cultures.">Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures.</a> Blood. 96 (9), 3029-3039 (2000).</li><li>Mayordomo, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow-derived+dendritic+cells+pulsed+with+synthetic+tumour+peptides+elicit+protective+and+therapeutic+antitumour+immunity.">marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.</a> Nature Medicine. 1 (12), 1297-1302 (1995).</li><li>Condon, C., Watkins, S. C., Celluzzi, C. M., Thompson, K., Falo, L. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strength+of+the+porcine+proximal+femoral+epiphyseal+plate:+The+effect+of+different+loading+directions+and+the+role+of+the+perichondrial+fibrocartilaginous+complex+and+epiphyseal+tubercle+-+An+experimental+biomechanical+study.">Strength of the porcine proximal femoral epiphyseal plate: The effect of different loading directions and the role of the perichondrial fibrocartilaginous complex and epiphyseal tubercle - An experimental biomechanical study.</a> Journal of Experimental Orthopaedics. 1 (1), 4 (2014).</li><li>Labeur, M. 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人类和小鼠具有不同的DC亚群，包括经典DC（cDC，包括cDC1和cDC2s），浆细胞样DC（pDC）和单核细胞衍生DC（MoDC）9，10，11。人们普遍认为，cDC1s调节细胞毒性T淋巴细胞（CTL）对细胞内病原体和癌症的反应，cDC2调节对细胞外病原体，寄生虫和过敏原的免疫反应12。在GM-CSF和IL-46，<sup class="...

树突状细胞（DC）是最强大的抗原呈递细胞（APC），用于激活幼稚的T细胞并诱导针对传染病，过敏性疾病和肿瘤细胞的特定细胞毒性T淋巴细胞（CTL）反应1，2，3。DC是先天免疫和适应性免疫之间的主要纽带，在免疫防御和维持免疫耐受性方面起着至关重要的作用。在过去的40年中，许多研究人员试图定义DC的亚群及其在炎症和免疫中的功能。根据这些研究，DC从骨髓细胞沿着骨髓和淋巴系发展。近年来，肿瘤疫苗取得了重大的里程碑，并拥有光明的未来。在机械上，肿瘤疫苗通过使用肿瘤抗原激活细胞毒性T淋巴细胞来调节免疫反应并防止肿瘤生长。基于DC的疫苗在肿瘤免疫治疗中起着重要作用，已被确定为最有前途的抗肿瘤疗法之一1，4。此外，DC已广泛应用于新型分子靶向药物和免疫检查点抑制剂的检测5.
研究人员迫切需要大量的高纯度DC来进一步研究DC的作用。然而，DC在各种组织和血液中很少见，仅占人类和动物血细胞的1%。骨髓树突状细胞（BMDC）的 体外 培养是获得大量DC细胞的重要方法。同时，用于从骨髓中生成DC的Lutz协议已被研究人员广泛使用6。虽然该方案在获得DC细胞方面是有效的，但它是复杂和昂贵的，涉及添加高浓度的细胞因子和红细胞的裂解。
在这项研究中，我们报告了一种从小鼠骨髓（BM）中分离几乎所有骨髓细胞的方法，并在 体外孵育7-9天后诱导分化为BMDC，GM-CSF和IL-4的浓度较低。该过程只需10分钟即可收获几乎所有骨髓细胞并将其悬浮在完整的培养基中。简而言之，我们在本研究中为BMDC提供了一种高效且具有成本效益的养殖方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Solarbio</td>
			<td>M8211</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-MHC II</td>
			<td>Biolegend</td>
			<td>116417</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10100</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CD80</td>
			<td>Biolegend</td>
			<td>104707</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Percp/cy5.5-CD11c</td>
			<td>Biolegend</td>
			<td>117327</td>
			<td></td>
		</tr>
		<tr>
			<td>PRMI-1640</td>
			<td>Thermo</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse GM-CSF</td>
			<td>Solarbio</td>
			<td>P00184</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-4</td>
			<td>Solarbio</td>
			<td>P00196</td>
			<td></td>
		</tr>
		<tr>
			<td>TruStain Fc PLUS (anti-mouse CD16/32) Antibody</td>
			<td>Biolegend</td>
			<td>156603</td>
			<td></td>
		</tr>
	</tbody>
</table>

economical and efficient protocol for isolating and culturing bone marrow-derived dendritic cells from mice

随着免疫学研究的进展，对树突状细胞（DC）的需求正在逐渐增加。然而，DC在所有组织中都很少见。分离DC的传统方法主要涉及通过注射大剂量（>10 ng / mL）的粒细胞 - 巨噬细胞集落刺激因子/ 白细胞介素-4（GM-CSF / IL-4）来诱导骨髓（BM）分化为DC，使该过程复杂且昂贵。在该方案中，使用在10ng / mL GM-CSF / IL-4培养基中培养的所有BM细胞，经过3-4次半培养交换后，每只小鼠（两个股骨）收获高达2.7 x 107 CD11c + 细胞（DC），纯度为80%-95%。培养10天后，CD11c，CD80和MHC II的表达增加，而细胞数量减少。培养7天后细胞数量达到峰值。此外，该方法仅需10分钟即可收获所有骨髓细胞，并且在培养1周后获得大量DC。

从两个股骨中提取1 x 10 7-1.7 x10 7个细胞，并在种植在6孔板中之前重新悬浮在24mL培养基中（图1A）。2天后，通过完全改变培养基去除非贴壁细胞。在更换培养基之前，观察到大量悬浮细胞（图1B）。培养3天后，小细胞集落开始形成。第六天，菌落的大小和数量显著增加。在第七天，细胞数量达到峰值（22 x 10 6-27 x 106），...

Watch this Scientific Journal Video about Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice at JoVE.com

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

在这里，我们提出了一种经济有效的方法，在用10 ng / mL GM-CSF / IL-4培养7天后从小鼠中分离和产生高纯度骨髓来源的树突状细胞。

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

从小鼠分离和生成骨髓衍生树突状细胞的经济有效的方案。涉及动物受试者的程序已获南京医科大学动物伦理委员会批准。随着肿瘤免疫研究的增加，对DC细胞的需求逐渐增加。然而，DC在所有组织中都很少见。以骨髓分化为DC为主的传统方法复杂且成本高昂。二。骨髓的分离和BM细胞的制备 我们处死小鼠并固定在小鼠手术台上。切除左侧暴露的肌肉和股动脉的皮肤。不要直接切除股动脉。否则，会引起大量出血并污染视野。切除股骨周围的所有肌肉。慢慢地将小鼠的下肢向外拉伸，直到股骨头脱垂，可以观察到。将下肢与身体分开。切开后腿以获得自由和完整的股骨。使用纱布去除肌肉。不要直接撕裂肌肉。小鼠的股骨很脆弱。将股骨置于75%酒精中两到五分钟。三。BMDC注射培养 用PBS冲洗残留的酒精。用止血镊子夹住股骨的中下部，用另一个止血钳夹住股骨的下端。止血钳从字面上施加到股骨上，股骨从骨骺线断裂。使用一ml注射器从骨骺线刺穿骨髓腔。使用完整的培养基两部分冲洗骨髓腔，直到骨头变白。收集冲洗液。补充含有GM CSF / IL-4至24ml的完整培养基，并在6孔板中播种，每孔4ml。四.结果.两天后，更换所有含有GM-CSF / IL-4的培养基。在第四天、第六天和第八天，一半人更换了含有GM-CSF / IL-4的完整培养基。细胞数量在第七天达到峰值，然后逐渐减少。DC细胞形成大量突触，呈现典型的成熟DC细胞形态。流量子指标分析表明，CD11c在第6天施加的比率为71%，在第10天将宽比提高到96.1%。CD11c、CD80和MSC2的表达随着培养时间的增加而逐渐增加。五、结论。简而言之，我们建立了一种经济有效的方案，用于从小鼠中分离和生成骨髓来源的树突状细胞，只需10分钟即可分离骨髓细胞。在培养6至7天后收获大量高纯度DC，每毫升10ng，GM-CSF和IL-4。

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

一种新的生理免疫原树突状细胞生成方法

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...