Name: isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method
Uploaded: 2022-07-13T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

这项工作得到了浙江省自然科学基金的支持（赠款编号：LY19H060001）和浙江省中医药科技计划项目（编号：2022ZB093）。

本研究的所有实验均按照浙江医科大学实验动物研究中心的动物实验指南（批准号：IACUC-20181029-11）进行。
1. 细胞提取
<ol><li>将Sprague-Dawley大鼠（SD大鼠，1-10天大，雄性或雌性）放入装有CO2 的安乐死笼中，以30%-70%的容器体积/分钟的平衡率。大鼠失去意识后（20-60分钟），通过宫颈脱位对大鼠实施安乐死，以确保无痛死亡。</li>
	<li>将大鼠浸泡在75%的酒精...

<ol>
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破骨细胞是参与骨病发生和发展的最重要细胞类型之一，也是骨病研究的主要对象之一20.单核细胞/巨噬细胞可分化为破骨细胞。由于单核巨噬细胞（RAW264.7细胞）购买成本太高，并且在培养过程中容易被激活，因此很难使用该细胞系进行 体外 分化实验。虽然已经开发了几种从骨髓中提取单核细胞/巨噬细胞的方法，包括密度梯度离心，胶原酶消化，微球富集和流式细胞术?...

据报道，存在于骨髓中的单核细胞-巨噬细胞系细胞可以分化成血液中的单核细胞和组织巨噬细胞1，2。上述细胞，其可分化成破骨细胞以平衡骨骼生长和发育，通常用作细胞模型以诱导体内破骨细胞3，4。骨髓是一种含有几种不同类型细胞的特殊组织，其包括但不限于骨髓间充质干细胞、骨髓单核巨噬细胞（BMM）、造血干细胞、内皮细胞和免疫细胞。事实上，之前的几项研究表明，从长骨的骨髓腔中冲出的贴壁细胞可以分化成成成骨细胞，破骨细胞，软骨细胞或脂肪细胞5，6，7，8。虽然已经使用了不同的分离和培养方法来产生不同的均质细胞群，但在从各种不同的细胞类型中分离和培养BMM仍然存在很大的挑战。
已经开发了几种方法来提取骨髓单核巨噬细胞（BMSC）。然而，这些方法中的大多数都是复杂的9，10，11。例如，密度梯度离心需要专门的试剂盒，操作既耗时又繁琐。该方法适用于从高容量血液样本中分离出BMM，但不适用于从骨髓样本9，12，13中分离。此外，使用胶原酶消化提取组织样品是一个复杂且耗时的过程;该方法不推荐用于从骨髓样本14，15中分离BMM。此外，尽管流动分离可以导致高度纯化的单核细胞/巨噬细胞群，但它需要非常大的样品量和高仪器设备要求10，16。此外，微珠富集法极其昂贵，并且在一般实验室17中是不可行的。
因此，在目前的研究中，提出了一种方便，快速，廉价的方法，用于从骨髓中分离单核巨噬细胞。使用不同时间点粘附的骨髓细胞通过二次粘附方法分离BMM。上述方法提取的BMM可以在 体外诱导破骨细胞的形成，从而为今后的 体外骨质疏松症研究提供简单方便的细胞模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm2 cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cathepsin K</td>
			<td>Abcam</td>
			<td>ab19027</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-CD11 isotype control</td>
			<td>Abcam</td>
			<td>ab172730</td>
			<td>1 &#956;g/test,1.675 mg/Ml</td>
		</tr>
		<tr>
			<td>Anti-CD11b/c</td>
			<td>Absin</td>
			<td>abs124232</td>
			<td>&#160;1&#956;g/test, 1 mg/mL</td>
		</tr>
		<tr>
			<td>Anti-TRAP</td>
			<td>Abcam</td>
			<td>ab191406</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin</td>
			<td>Beyotime</td>
			<td>&#160;AF5003</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td>for IF, 1:2,000</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td>for WB, 1:2,000</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Pepro tech</td>
			<td>400-28</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biosharp</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>Pepro tech</td>
			<td>400-30</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rat</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd</td>
			<td></td>
			<td>1-10 days old</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel preparation kit</td>
			<td>Solarbio</td>
			<td>P1200</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP/ALP Staining Kit</td>
			<td>Wako</td>
			<td>294-67001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Biosharp</td>
			<td>BL512A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright-Giemsa solution</td>
			<td>Keygen Biotech</td>
			<td>KGA225-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method

随着骨矿物质密度的降低，骨骼更容易骨折，从而对患者的生活质量产生负面影响。骨骼的生长发育主要由成骨细胞和破骨细胞调节。人们普遍认为破骨细胞来源于骨髓单核细胞-巨噬细胞（BMM）。BMM和其他造血干细胞位于骨髓腔内。因此，从不同和异质细胞群中分离单个稳定的BMM是一个巨大的挑战。在这里，我们提出了一种从SD大鼠中分离BMM的方案，称为二次依从性方法。收集在原代培养物中培养24-48小时的贴壁细胞。流式细胞术分析显示，约37.94%的细胞为CD11b/c+（单核细胞-巨噬细胞表面抗原）。酒石酸盐耐酸磷酸酶（TRAP）染色和蛋白质印迹分析表明，BBM 可以在体外分化为破骨细胞。上述结果表明，二级依从性细胞可被视为破骨细胞分化研究的合适细胞模型。

次级贴壁细胞群稳定且均匀。随着细胞的连续增殖，大多数细胞变得更大，形状不规则并生长成径向贴壁盘（图2C，D）。流式细胞术显示，表达CD11b / c（单核巨噬细胞谱系细胞表面的分子标记物）的细胞百分比约为37.94%（图2A，B）。为了进一步验证CD11b / c阳性细胞是单核细胞 - 巨噬细胞谱系细胞，在用...

Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

在这里，我们提出了一种从SD大鼠中分离BMM的方案，称为二次依从性方法。

通过二级粘附法从大鼠骨骼中分离单核细胞 - 巨噬细胞谱系细胞

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

在该方案中，通过二级依从性方法提取骨髓单核细胞 - 巨噬细胞。骨髓单核细胞 - 巨噬细胞可以成功分化为破骨细胞，这为体外破骨细胞的研究提供了稳定的细胞模型。该方法适用于小骨髓样品，无需高科技实验室设备即可简单快速地从骨髓中提取骨髓单核细胞 - 巨噬细胞。破骨细胞的增加可能是骨质疏松症的潜在发病机制。骨髓单核细胞-巨噬细胞作为破骨细胞前体细胞具有一定的研究价值。首先，将安乐死的大鼠浸泡在75%的酒精中10分钟进行消毒。用剪刀和镊子小心地取出大鼠的所有四肢后，用移液管用PBS吸出并冲洗粘附在四肢上的血液。然后在两毫升的10%FBS DMEM中放入一个五毫升的管中。将肢体骨转入五毫升管中后，用剪刀将管中的肢体骨切成小块，混合匀浆，将骨髓细胞重悬于培养基中。静置五分钟，直到组织碎片沉降到管的底部。接下来，将10毫升10%FBS DMEM加入100毫米培养皿中，然后将上清液转移到培养皿中。在37摄氏度的5%二氧化碳中孵育约24小时。在此孵育时间之后，大多数间充质干细胞将粘附在培养皿壁上并缓慢生长，而大多数骨髓单核细胞 - 巨噬细胞仍将悬浮在培养基中。将100毫米培养皿中的细胞悬浮液转移到新的25厘米见方的烧瓶中，并继续在37摄氏度的5%二氧化碳中培养细胞24小时。小心取出旧培养基后，在骨髓单核细胞 - 巨噬细胞粘附在烧瓶壁上后，用新鲜培养基替换。流式细胞术显示，表达CD11b和c（单核细胞-巨噬细胞谱系细胞表面的分子标志物）的细胞比例约为37.94%，随着细胞的连续增殖，大多数细胞在不规则形状内变大并生长成径向贴壁盘。TRAP染色结果显示，与对照组相比，用核因子kappa B配体受体激活剂和巨噬细胞集落刺激因子诱导的细胞内紫红色颗粒数量显著增加。蛋白质印迹分析表明，与对照组相比，处理细胞中破骨细胞特异性蛋白TRAP和组织蛋白酶K的表达水平显着增加。在这个过程中最重要的是收集次级贴壁细胞的时间在24至48小时的原代培养物中，骨髓单核细胞巨噬细胞开始完全粘附。通过该方法分离的骨髓单核细胞 - 巨噬细胞可以在体外诱导成破骨细胞，为骨质疏松症的研究提供稳定的细胞模型。

Research

JoVE Journal

Medicine

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型：教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

足月胎盘胎膜中原发性人蜕细胞的分离

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

代谢综合征心力衰竭模型大鼠心房心肌细胞的分离与保留射血分数的关系

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...