资助：本研究由国家自然科学基金资助（第82004154、81900311、82100336、81970345号）资助。

该研究是在四川大学华西医院动物伦理委员会授予的项目许可（编号20211404A）下进行的，符合四川大学华西医院动物伦理委员会关于动物护理和使用的指南。根据伦理准则，本研究中使用的大鼠保持在受控环境中，光/暗循环为12小时，温度为22-24°C，湿度为50%-60%。老鼠可以随意获得食物和水。本研究中使用的动物是 6-8 周龄的 Sprague Dawley （SD） 雄性大鼠，体重约 250 g，无特定病原体。这些动物是从商业来源获得的（见 材料表）。
1.大鼠中性粒细胞的分离
<ol><li>骨髓采集<ol><li>将大鼠置于适当的容器中进行麻醉（见 材料表）。给大鼠施用3%异氟醚，直到动物失去知觉。检查是否对脚趾捏或尾巴捏没有反应，以确保麻醉深度。</li>
		<li>一旦大鼠被深度麻醉，通过将大鼠放在其背上并用一只手握住它的尾巴，同时用另一只手抓住头部来进行颈椎脱位。通过对尾巴施加突然而强大的向上力，同时向下拉动头部，直到颈椎被切断，从而快速而牢固地使颈部脱臼。等待确认死亡，例如停止呼吸和心跳不畅，然后再进行组织收集或处置。</li>
		<li>将大鼠浸入75%乙醇中，静置几分钟，对皮毛和皮肤进行消毒。将大鼠仰卧并切断臀部的下肢以保护股骨头。用解剖剪刀切开膝关节处的韧带，将膝关节向后折叠，露出股骨和胫骨。</li>
		<li>使用镊子和剪刀，小心地去除任何与骨骼相连的肌肉、肌腱和其他组织。用 Hank 平衡盐溶液 （HBSS） 冲洗骨骼，以去除任何残留的组织碎片和血液。再重复两次，总共洗涤三次。将清洗干净的骨头放入无菌容器中。</li>
		<li>使用 5 或 10 mL 注射器和针头刺穿骨髓两端，使骨髓基质松弛。用另一个注射器用 10-15 mL 罗斯威尔公园纪念研究所 （RPMI） 培养基冲洗骨髓，直到没有可见的骨髓可以冲洗掉。</li>
		<li>在20°C下以300× g 离心骨髓细胞10分钟。 加入 5-10 mL 红细胞裂解缓冲液（参见 材料表）重悬细胞并在室温下孵育 3 分钟。向混合物中加入 4 倍体积的 HBSS。在室温下以600× g 离心细胞5分钟，用移液管弃去上清液，并使用所得沉淀进一步使用。</li>
	</ol></li>
	<li>从骨髓中分离中性粒细胞<ol><li>对于两步法，请执行以下附加步骤：<ol><li>按照制造商的说明使用商业化的大鼠中性粒细胞分离试剂盒分离骨髓细胞（参见 材料表）。</li>
			<li>通过在新的 15 mL 离心管中分层 2 mL 55% percoll 试剂（参见 材料表）、2 mL 65% 试剂、2 mL 70% 试剂和 2 mL 80% 试剂来制备密度梯度。在20°C下以800× g 离心管40分钟，加速度设置为5，减速设置为0或1。</li>
			<li>使用无菌移液管收集70%梯度层，包括65%和70%梯度试剂边界处的细胞层。将细胞悬液转移到新的 15 mL 离心管中。将 15 mL HBSS 加入试管中，轻轻倒置试管数次以洗涤细胞。在室温下以300× g 离心管10分钟以沉淀细胞。</li>
		</ol></li>
		<li>对于一步法，请执行以下附加步骤：<ol><li>在不使用大鼠中性粒细胞分离试剂盒的情况下将骨髓细胞置于梯度下。</li>
			<li>使用无菌移液管收集70%梯度层，包括65%和70%梯度之间边界的细胞层。将细胞悬液转移到新的 50 mL 离心管中，并用 HBSS 洗涤细胞。在室温下以400× g 离心管5分钟以沉淀细胞。</li>
			<li>使用血细胞计数器计数分离的中性粒细胞，并使用台盼蓝染色评估活力。 注意：可以根据大鼠的数量和所需的分离中性粒细胞数量修改方案。当这种方法首次在新的实验环境中使用时，从三只大鼠身上获得了可接受的骨骼量。所有程序都应在无菌条件下进行。应尽快从骨髓中分离中性粒细胞，以尽量减少细胞损伤和活力丧失。</li>
		</ol></li>
	</ol></li>
</ol>2. 大鼠神经内分泌瘤的获取
<ol><li>在无菌的10cm培养皿中，将0.5×108分离的中性粒细胞重悬于4mLRPMI培养基（补充有10%胎牛血清（FBS）和1%青霉素 - 链霉素）中。</li>
	<li>将500nM PMA（参见 材料表）添加到中性粒细胞悬浮液中以诱导NETosis。在37°C和5%CO2下孵育3小时。</li>
	<li>对于阴性对照，将 DNase I （10 U/mL） 加入中性粒细胞悬浮液中以降解分泌的 NET。</li>
	<li>要收获 NET，请取出培养基并用 HBSS 轻轻清洗附着在培养皿上的 NET。然后，对每个板使用 4 mL 新鲜培养基进行强烈冲洗，以将 NET 从板上分离。</li>
	<li>经常收集洗涤介质和移液管，以完全重悬 NET。</li>
	<li>将悬浮液在20°C下以300× g 离心10分钟以除去任何漂浮的细胞。</li>
	<li>将含有NET的悬浮液转移到无菌管中，并储存在-20°C下，以便在2周内进一步使用。 注意：NET对降解很敏感，因此建议使用新鲜收获的材料以获得最佳效果。</li>
</ol>3. 验证是否存在神经内分泌瘤
<ol><li>将细胞（从步骤2.4开始）固定在带有4%多聚甲醛的盖玻片上15分钟，将NET（从步骤2.7开始）固定30分钟至1小时。</li>
	<li>将盖玻片倒置在石蜡膜覆盖的试管架上的PBS / PBST液滴上，并重复洗涤过程三次，每次5分钟。</li>
	<li>在室温下用0.5%Triton-X-100透化细胞10分钟，并在PBS / PBST中洗涤3次，每次1分钟。用10%正常驴血清（参见 材料表）在室温下密封细胞1小时。</li>
	<li>将细胞与抗大鼠中性粒细胞弹性蛋白酶抗体和抗大鼠髓过氧化物酶抗体（参见 材料表）在4°C下孵育过夜。 然后，在PBS / PBST中洗涤盖玻片三次，每次5分钟。</li>
	<li>将细胞与二抗A488偶联的驴抗兔IgG（H + L），A594偶联的驴抗小鼠IgG（H + L）和A594偶联的山羊抗小鼠IgG1（参见 材料表）在室温下在黑暗中孵育1小时。然后，在PBS / PBST中洗涤盖玻片三次，每次5分钟。</li>
	<li>用 10 mg/mL DAPI 染色细胞核和 NET 骨架。然后，在PBS / PBST中洗涤盖玻片三次，每次5分钟。</li>
	<li>将一滴封固剂放在载玻片上，然后将带有细胞的盖玻片倒置在其上。让它干燥1小时，用浸入式透镜进行显微镜分析;否则，它就可以进行检查了。 注意：操作NET时必须格外小心，即使在固定后也是如此，因为它们非常脆弱，在准备过程中很容易丢失。</li>
</ol>4. NETs的定量
<ol><li>根据制造商的说明制备Tris-EDTA（TE）缓冲液和PicoGreen（dsDNA测定试剂）工作溶液（参见 材料表）。</li>
	<li>通过将储备 DNA 稀释至 1 ng/mL、10 ng/mL、100 ng/mL 和 1 μg/mL 的浓度来制备标准品。 注意：为了量化NET的浓度，采用了dsDNA检测试剂盒（见 材料表），并遵循制造商的指南。分别使用 19 倍体积的双蒸水或 199 倍体积的 TE 缓冲液制备 Tris-EDTA （TE） 缓冲液和 dsDNA 检测试剂。随后，制备标准溶液（1 ng/mL、10 ng/mL、100 ng/mL 和 1 μg/mL），并将每 50 μL 样品与 450 μL TE 缓冲液混合。</li>
	<li>将每个样品的 50 μL 加入 450 μL TE 缓冲液中。</li>
	<li>向 96 孔板中的每个孔中加入 100 μL 标准品或样品以及等体积的测定试剂工作溶液，包括标准品和样品。</li>
	<li>将板在室温下孵育2-5分钟，避免直射。</li>
	<li>使用发射光谱约为 530 nm、吸光度光谱约为 480 nm 的荧光酶标仪读取样品。</li>
	<li>使用标准品生成的标准曲线计算每个样品中NET的浓度。</li>
</ol>5. 细胞细胞术分析NETs分泌
<ol><li>将细胞核染色剂（参见 材料表）添加到在96孔板中孵育的细胞中，以达到10ng / mL的终浓度并孵育30分钟。</li>
	<li>向细胞中加入游离DNA（cfDNA，参见 材料表）染色剂，以达到300nM的终浓度并孵育10分钟。</li>
	<li>通过轻柔的移液混合荧光染料。不要丢弃上清液或洗涤沉淀。</li>
	<li>将样品板装入细胞仪中。</li>
	<li>设置不同通道的对焦和曝光时间参数：蓝色（例如：377/50 nm，em：470/22 nm），通常曝光时间为 30,000 毫秒，绿色（例如：483/32 nm，eM：536/40 nm），通常曝光时间为 5,000 毫秒。</li>
	<li>在不同通道下捕获整个板的高度均匀图像。</li>
	<li>分析不同组的细胞核染色计数。如果它们相似，则可以通过cfDNA染色计数来确定NETs的分泌。</li>
</ol>

高效的大鼠骨髓中性粒细胞胞外陷阱分离

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作者没有利益冲突需要声明。

中性粒细胞的分离是研究NETosis的关键步骤，其中选择适当的分离方法对于获得可靠的结果至关重要。需要权衡的一个重要因素是分离过程中淋巴细胞污染的发生。在从骨髓中分离大鼠中性粒细胞时，应对这一挑战尤为重要。尽管中性粒细胞（1.0814-1.0919，峰值为1.0919）与淋巴细胞（1.0337-1.0765，峰值为1.0526）相比具有不同的密度范围，但淋巴细胞污染仍然不可避免。这在一定程度上可以归因于骨髓?...

中性粒细胞是白细胞的一个关键亚群，在先天免疫反应中起着关键作用。它们的特征是含有各种蛋白酶和抗菌肽的多叶细胞核和颗粒1。中性粒细胞主要通过脱颗粒、吞噬作用和神经内分泌瘤的形成发挥作用。1996 年，Takei 等人在用肉豆蔻酸佛波醇酯 （PMA） 刺激中性粒细胞的实验中首次观察到了 NET。随后，Brinkmann 等人 3 在 2004 年创造了 NET 形成过程“NETosis”。他们的研究进一步阐明了NETs在中性粒细胞介导的抗菌反应中的关键作用。NET是由染色质、组蛋白和抗菌蛋白组成的网状结构，这些蛋白在感染和炎症刺激下从活化的中性粒细胞中释放出来。NET可以通过捕获入侵的病原体并将其暴露于高浓度的抗菌肽和蛋白酶来固定和杀死入侵的病原体1,3。此外，神经内分泌瘤有助于细胞凋亡的清除并参与炎症消退。最近的研究还表明，神经内分泌瘤的过度形成或神经内分泌瘤降解受损可导致组织损伤、自身免疫性疾病、血栓形成和血运重建受损4,5,6,7,8,9,10。
NETs在心肌梗死后不受控制的纤维化和室性动脉瘤形成中的致病作用已通过血管周围纤维化的扩展得到证实4,11。心肌梗死模型和从小鼠骨髓中分离中性粒细胞都是公认的。多形核 （PMN） 白细胞是一种富含人体血液的白细胞，是分离人类中性粒细胞的极好来源。这种方法消除了采集骨髓的需要，从而提高了安全性和效率。
神经内分泌瘤在与心脏重塑相关的心房颤动中也起作用。然而，大型动物（如狗和猪）被用来模拟心房颤动，因为小鼠缺乏足够大的心房来建立折返周期或AF模型，除非特定的离子通道或信号通路被敲低或敲除12。虽然如前所述，可以在大鼠中诱导心房颤动并从大鼠外周血中分离中性粒细胞，但研究人员遇到了一个限制，即只能从外周血中分离出 2 x 105-5 x 105 中 性粒细胞（每只大鼠 10 mL）。在每个时间点提取足够的 NET 需要大约 10-25 只大鼠（总共 5 x 106 个中性粒细胞），导致耗时、昂贵且通常产量低的过程13。在这方面，Li He及其同事提出了一种以骨髓为导向的策略，以从大鼠中获得足够的NETs14。在他们的文章中，他们全面描述了从大鼠骨髓中分离中性粒细胞，并比较了大鼠外周和骨髓中性粒细胞的NET分泌能力。概述的两种方法迎合了不同的实验目标，都产生了足够数量的大鼠骨髓中性粒细胞，同时减少了所需大鼠的数量。两步分离法的中性粒细胞纯化性能优异，而一步法则在可接受的纯化水平下具有时间效率。此外，研究人员比较了大鼠骨髓中性粒细胞与其外周对应物之间的NETosis和NET形成，发现与PMN的效力相同。这些发现对心房颤动的中性粒细胞相关研究做出了重大贡献，并强调了在具有不同中性粒细胞分布的各种实验动物中灵活选择不同来源进行中性粒细胞分离的重要性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A488-conjugated donkey antirabbit IgG(H + L)</td>
			<td>Invitrogen, USA</td>
			<td>A32790</td>
			<td></td>
		</tr>
		<tr>
			<td>A594-conjugated donkey anti-mouse IgG(H + L)</td>
			<td>Invitrogen, USA</td>
			<td>A32744</td>
			<td></td>
		</tr>
		<tr>
			<td>A594-conjugated goat anti-Mouse IgG1&#160;</td>
			<td>Invitrogen, USA</td>
			<td>A21125</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat myeloperoxidase</td>
			<td>Abcam, England</td>
			<td>ab134132</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat neutrophil elastase</td>
			<td>Abcam, England</td>
			<td>ab21595</td>
			<td></td>
		</tr>
		<tr>
			<td>Celigo Image Cytometer</td>
			<td>Nexelom, USA</td>
			<td>200-BFFL-5C</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma, USA</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>Gibco, USA</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco, USA</td>
			<td>C14175500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Thermofisher, USA</td>
			<td>33342</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD, China</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Sigma, USA</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio, China</td>
			<td>SL050</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>biosharp, China</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Hyclone, USA</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE, USA</td>
			<td>P8370-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, USA</td>
			<td>&#160;P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Picogreen dsDNA Assay Kit</td>
			<td>Invitrogen, USA</td>
			<td>P11496</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat neutrophil isolation kit</td>
			<td>Solarbio, China</td>
			<td>P9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Solarbio, China</td>
			<td>R1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) media</td>
			<td>Hyclone, USA</td>
			<td>SH30809.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>RWD Universal Animal Anesthesia Machine</td>
			<td>RWD, China</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley (SD) rats</td>
			<td>Dashuo, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SytoxGreen</td>
			<td>Thermofisher, USA</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td>Solarbio, China</td>
			<td>T1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Biofroxx, German</td>
			<td>1139ML100</td>
			<td></td>
		</tr>
	</tbody>
</table>

unique approach for isolating rat bone marrow neutrophils with comparable capacity

这项研究的主要目的是开发一种可靠有效的方法，用于从大鼠骨髓中分离中性粒细胞胞外陷阱 （NET）。这种努力是由于与从外周血中提取NET的传统方法相关的局限性而产生的，主要是由于可用于分离的中性粒细胞稀缺。该研究揭示了从骨髓中获取大鼠中性粒细胞的两种不同方法：一种是简化的一步法，可产生令人满意的纯化水平，另一种是更耗时的两步法，其纯化效率更高。重要的是，这两种技术都产生了大量的活中性粒细胞，每只大鼠在5000万到1亿之间。这种效率反映了从人类和小鼠来源分离中性粒细胞所获得的结果。值得注意的是，与从外周血中获得的中性粒细胞相比，来自大鼠骨髓的中性粒细胞表现出相当的分泌NET的能力。然而，基于骨髓的方法始终产生大量中性粒细胞和神经内分泌瘤。这种方法证明了获得更多数量的这些细胞成分用于进一步下游应用的潜力。值得注意的是，这些分离的神经内分泌瘤和中性粒细胞有望用于一系列应用，涵盖炎症、感染和自身免疫性疾病领域。

本文概述的方案描述了两种不同的方法，每种方法都以改进的纯化或简化的步骤为特征。两种方法每只大鼠产生约0.5 x 108-1 x 108 中性粒细胞。采用膜联蛋白 V-FITC/PI 细胞凋亡检测试剂盒进行流式细胞术分析，细胞活力超过 90%，与小鼠和人类细胞活力相当（图 1）。虽然在从骨髓中分离中性粒细胞的过程中，淋巴细胞污染似乎是不可避免的，但与一步法的 50% ?...

Watch this Scientific Journal Video about Author Spotlight: Optimized Method for Isolating Rat Neutrophils and NETs from Bone Marrow at JoVE.com

作者聚焦：从骨髓中分离大鼠中性粒细胞和 NET 的优化方法

这项研究概述了从大鼠骨髓中分离大量中性粒细胞胞外陷阱 （NET） 的两种技术。一种方法将商用中性粒细胞分离试剂盒与密度梯度离心相结合，而另一种方法仅采用密度梯度离心。这两种方法产生的功能性神经内分泌瘤都超过了外周血中性粒细胞。

分离具有相当容量的大鼠骨髓中性粒细胞的独特方法

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone ...

我们开发了一种分离大鼠中性粒细胞并随后提取 NET 的方法。该方法优化了在大鼠和大鼠来源细胞系中进行 NET 相关实验的可及性。在我们的研究中，我们对从外周血和骨髓线中分离的中性粒细胞进行了比较。我们的研究结果强调了骨髓来源的中性粒细胞的优越性，展示了分离的中性粒细胞计数的巨大数量优势以及它们熟练的同步能力。我们基于骨髓分离大鼠中性粒细胞和神经内分泌瘤的方法通过优化这些细胞类型的分离技术填补了研究空白。与以前主要使用外周血的方法不同，我们的方案靶向骨髓，以高产量增加中性粒细胞，并为研究中性粒细胞和大鼠生物学提供更准确的大鼠模型。

unique-approach-for-isolating-rat-bone-marrow-neutrophils-with

Research

JoVE Journal

Biology

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

1.7K 次观看.  Sichuan University. 我们开发了一种分离大鼠中性粒细胞并随后提取 NET 的方法。该方法优化了在大鼠和大鼠来源细胞系中进行 NET 相关实验的可及性。在我们的研究中，我们对从外周血和骨髓线中分离的中性粒细胞进行了比较。我们的研究结果强调了骨髓来源的中性粒细胞的优越性，展示了分离的中性粒细胞计数的巨大数量优势以及它们熟练的同步能力。我们基于骨髓分离大鼠中性粒细胞...

视频: 作者聚焦：从骨髓中分离大鼠中性粒细胞和 NET 的优化方法

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

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

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

表型鼠标肺功能<em&gt;在体内</em&gt;与肺弥散功能

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

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

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

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

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

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

小鼠新鲜骨髓外植体的丙酸盐形成动力学

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...