这项工作得到了美国国立卫生研究院 [R01 AI163333] 对 CMR 的支持。我们感谢以下赠款为西弗吉尼亚大学流式细胞术和单细胞核心设施提供的额外资金支持：WV CTSI 赠款 GM104942、肿瘤微环境 CoBRE 赠款 GM121322 和 NIH 赠款 OD016165。

所有程序均由西弗吉尼亚州机构动物护理和使用委员会批准，并按照国家研究委员会的《实验动物护理和使用指南》的建议进行。本研究使用 C57BL/6J 小鼠幼崽。材料 表中列出了所有使用的试剂和设备的详细信息。
1. 培养基制备
<ol><li>在 5 mL 离心管中制备 3 mL 补充有 10% FBS、2 mM 谷氨酰胺、25 mM HEPES 和青霉素 （100 U/mL）/链霉素 （100 μg/mL） 的 MEM 培养基，并保持在冰上。</li>
	<li>如果需要将骨髓祖细胞储存在液氮中，请准备含有 10% DMEM、80% FBS 和 10% DMSO 的冷冻培养基。</li>
	<li>对于完整的 DMEM，制备补充有 10% FBS、2 mM 谷氨酰胺、25 mM HEPES 和青霉素 （100 U/mL）/链霉素 （100 μg/mL） 的 DMEM。</li>
	<li>对于巨噬细胞分化，制备补充有 10% FBS、2 mM 谷氨酰胺、青霉素 （100 U/mL）/链霉素 （100 μg/mL） 和 10% L929 细胞上清液的 DMEM9,10,11。</li>
</ol>2. L929 细胞上清液制备
<ol><li>解冻储存在液氮中的 L929 细胞，并转移至含有 5 mL 完整 DMEM 的 15 mL 离心管中。在室温下以 350 x g 离心 5 分钟。</li>
	<li>将细胞重悬于 5 mL 完全 DMEM 培养基中，并以 2×105 个细胞的密度将它们接种到 T25 组织培养瓶中。使用 0.4% 台盼蓝的自动细胞计数器对细胞进行计数。在 37 °C 下用 5% CO2 孵育，直到它们达到 70% 汇合。</li>
	<li>要分离细胞，首先用 5 mL 磷酸盐缓冲盐水 （PBS） 洗涤它们。吸出 PBS 并用 2.5 mL 0.05% 胰蛋白酶-EDTA 替换。</li>
	<li>在 37 °C 下用 5% CO2 孵育 5 分钟，然后加入 5 mL 完整的 DMEM。</li>
	<li>收集细胞并在室温下以 350 x g 离心 5 分钟。</li>
	<li>将细胞沉淀重悬于 15 mL 完整 DMEM 培养基中，并将其转移到 T75 培养瓶中。</li>
	<li>在 37 °C 和 5% CO2 下孵育，直到细胞达到 90% 汇合，然后收集培养基并在 4 °C 下以 500 x g 离心 5 分钟。 通过 0.45 μm 过滤器过滤含有 M-CSF 的培养基，并在 -80 °C 下以 5 mL 等分试样冷冻。</li>
</ol>3. 动物准备
<ol><li>在生物安全柜下，小心地将 7-9 天大的 C57BL/6J 小鼠幼崽（一窝 6 只幼崽）与母鼠分开，并将它们放在单独的笼子中。</li>
	<li>将棉球浸泡在钟罩或其他密封室中的 2 mL 兽用级异氟醚中。</li>
	<li>将一只小狗放入腔室并盖上盖子。监测新生儿约 90 秒，以确保其变得一动不动和失去知觉。</li>
	<li>在幼犬恢复意识之前，迅速取出幼犬并用锋利的剪刀（遵循机构批准的协议）将其斩首。 注意：新生儿仅通过吸入异氟醚呼吸不足以进行安乐死。</li>
	<li>确保在取出每只幼崽后盖上容器的盖子。同一个棉球可用于 5-7 只幼崽。</li>
</ol>4. 新生儿骨髓的分离
<ol><li>用 70% 乙醇对新生儿的身体进行消毒。</li>
	<li>“使用镊子和细尖手术剪刀，在 腹部和后腿。向后肢的脚部拉动，去除皮肤。</li>
	<li>使用锋利的镊子刮擦附着在骨骼上的结缔组织和肌肉。</li>
	<li>剪断胫骨和股骨，如图 1 所示，用剪刀分离和移除。使用镊子将这些骨头放入冰上的培养基管中。对每只小狗重复该过程。</li>
	<li>在镊子的帮助下将骨骼转移到带有收集管的 40 μm 过滤器中。用 3 mL 注射器柱塞压迫过滤器，压碎骨头并释放骨髓。</li>
	<li>将细胞悬液在 4 °C 下以 350 x g 离心 5 分钟。</li>
	<li>吸出上清液，并通过在 0.2% NaCl 中轻轻移液重悬细胞，NaCl 是一种用于裂解红细胞的低渗溶液。立即加入等体积的 1.6% NaCl，使溶液达到等渗。</li>
	<li>在 4 °C 下以 350 x g 离心 5 分钟，然后去除裂解液。</li>
	<li>重悬于完整的 DMEM 中以立即使用或冷冻培养基以储存，并使用血球计数器或自动细胞计数仪对细胞进行计数。 注意：对于本研究，该方法产生了 6.55 （± 1.44） × 106 个骨髓细胞/幼崽（图 1E）。</li>
	<li>将细胞储存在 -80 °C 的冷冻培养基中或立即使用，如下所述。 注意：由于年代久远，骨骼足够透明，可以通过它们看到骨髓。拉动骨头时要小心，因为对它们施加轻微的压力可能足以释放骨髓。新生儿骨骼中的骨髓是液体形式，而不是成人骨骼中的完整线。如果骨髓在收集骨骼过程中逸出，则很难收集骨髓。</li>
</ol>5. 新生儿骨髓来源的巨噬细胞的分化
<ol><li>将每个 T75 培养瓶× 2 7个骨髓细胞 接种在 10 mL 含有 10% L929 细胞上清液 （2.5 mL） 作为 M-CSF 来源的完整 DMEM 中。将培养皿在 37 °C 和 5% CO2 下孵育 5 天。在分化第 3 天加入 2 mL L929 条件培养基。</li>
	<li>每天使用倒置显微镜和放大倍数为 20 倍的成像系统在显微镜下观察细胞。分化后，巨噬细胞应显得粘附、细长和异质性（图 2）。</li>
	<li>要收获巨噬细胞用于下游分析，请吸出分化培养基。</li>
	<li>用 5 mL PBS 洗涤细胞，以去除会干扰脱离的非贴壁细胞和血清蛋白。吸出 PBS。</li>
	<li>向培养瓶中加入 5 mL 0.05% 胰蛋白酶-EDTA 来收获细胞。将培养瓶置于 37 °C 和 5% CO2 培养箱中 5 分钟，并加入 5 mL DMEM。</li>
	<li>移液细胞以分离并在室温下以 350 x g 离心 5 分钟。</li>
	<li>在 1 mL 完整的 DMEM 中解离细胞沉淀，并使用台盼蓝计数并检查细胞活力。使用这种方法分离的 BMDM 为 7.05 （± 2.52） ×105 个细胞/幼崽（图 1E）。 注意：观察在分化第二天开始出现的纺锤形细胞的存在（图 1B，红色箭头）。如果培养基的颜色变为黄色，表明它已用完，请在分化的第 4 天添加更多 L929 条件培养基;建议不要完全更换介质。</li>
</ol>6. 免疫标记和流式细胞术分析
<ol><li>根据制造商的建议，用 10 μL/107 个细胞的 FcR 封闭试剂在 100 μL/标签的流式细胞术缓冲液（PBS 补充有 2 mM EDTA 和 0.5% 牛血清白蛋白）中处理，阻断 BMDM 的非特异性抗体标记（2x105/标签）。在冰上保持 10 分钟。 注：细胞可以散装封闭，也可以单独封闭在单独的孔或试管中。所有后续步骤均指示最终体积为 100 μL 时每个细胞标记物的量和体积。</li>
	<li>加入 200 μL 流式细胞术缓冲液洗涤细胞，并在室温下以 525 x g 离心 7 分钟。</li>
	<li>去除上清液，将细胞以每孔 5 × 105 个细胞的密度接种在 U 形底 96 孔板中。通过在 100 μL 流式细胞术缓冲液中单独或组合加入稀释至 3,000 倍的 FVS780-活/死细胞计数染料和 0.625 μL BV786-CD11b、PE-F4/80 和 AlexaFluor488-CD206 对细胞进行染色 12,13,14,15。将细胞在覆盖有箔纸的冰上孵育 45 分钟。</li>
	<li>加入 100 μL 流式细胞术缓冲液，并在室温下以 525 x g 离心 7 分钟洗涤细胞。</li>
	<li>吸出上清液并轻轻涡旋细胞沉淀。向每个孔中加入 100 μL 0.4% 多聚甲醛，并在 4 °C 下孵育过夜。</li>
	<li>加入 100 μL 流式细胞术缓冲液洗涤细胞，并在室温下以 525 x g 离心 7 分钟沉淀。</li>
	<li>将细胞重悬于 400 μL 流式细胞术缓冲液中，并使用流式细胞仪进行分析。</li>
</ol>7 . 体外 试验评价新生儿骨髓来源的巨噬细胞的吞噬功效
<ol><li>将 BMDM 重悬于不含酚红或抗生素的完整 DMEM 中，并以 2 ×10 5/象限的密度将它们接种在 35 mm 四联培养皿中，体积为 500 μL。</li>
	<li>为了制备感染复数 （MOI） 为 25 或其他所需 MOI 的细菌接种物，请取出储存在 -80 °C 下的预先计算的大 肠杆菌 O1：K1：H7 原液。</li>
	<li>将所需体积的细菌分装到 1.7 mL 微量离心管中。通过添加 PBS 至总体积为 1 mL 来洗涤细菌。通过在室温下以 2,000 x g 离心 5 分钟来沉淀细菌。重复洗涤步骤。</li>
	<li>将细菌沉淀重悬于 50 μL PBS 中，并添加绿色荧光 pH 敏感染料至终浓度为 500 μM。这是一种对 pH 值敏感的染料，在中性 pH 值下没有荧光信号，在吞噬过程中，在酸性环境（如酸化吞噬体）中会发出明亮的荧光。</li>
	<li>将细菌细胞在黑暗中孵育 20 分钟以进行染料缀合。</li>
	<li>用 1 mL PBS 洗涤细菌四次，在室温下以 1000 × g 离心 5 分钟。</li>
	<li>将细菌重悬于 500 μL 不含酚红的完整 DMEM 中，并添加到 BMDM 培养物中。</li>
	<li>将 BMDM 在 37 °C 下在 5% CO2 培养箱中孵育 4 小时，然后加入 200 ng 细胞渗透性红色荧光染料，对溶酶体进行染色。 注意：吞噬细菌可以通过荧光显微镜观察。</li>
</ol>

新生儿骨髓来源巨噬细胞的吞噬试验评价 

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涉及新生小鼠模型的研究可能会带来许多挑战。与成人相比，新生儿的免疫系统发育中是独一无二的8。因此，不应假设从成年动物模型生成的数据适用于新生儿，几篇已发表的著作已经很好地阐明了这一想法18,19。因此，新生儿特异性模型和细胞来源对于研究早期免疫反应的复杂性是必要的。然而，由于新生小鼠的大小、敏感性和脆?...

骨髓包含造血干细胞群和间充质干细胞群，这些细胞群是自我更新的，可以分化成各种细胞谱系。骨髓中的造血干细胞产生骨髓和淋巴谱系1。间充质干细胞产生成骨细胞（骨骼）、脂肪细胞（脂肪）或软骨细胞（软骨）2。这些细胞在细胞生物学和组织工程领域有多种应用，包括基因治疗 3,4。存在于骨髓中的祖细胞在谱系特异性生长因子存在的情况下分化成特定的细胞类型。促红细胞生成素促进红细胞祖细胞的增殖，粒细胞集落刺激因子 （G-CSF） 刺激中性粒细胞集落的生长，血小板生成素调节血小板的产生，这是谱系特异性生长因子的几个例子5。细胞表面抗原标记的 FACS 和磁激活细胞分选 （MACS） 是分离和纯化特定骨髓来源细胞类型的成熟方法6。
尽管新生儿研究正在朝着寻找新生儿死亡原因和解决早产并发症的方向发展，但直接治疗开发仍然是一个未得到满足的医疗需求。Smith 和 Davis 表示，“儿科患者仍然是治疗性孤儿”7。在新生儿临床研究中，获得同意存在一些挑战，例如样本小、结果的终生影响以及获得同意的伦理问题8.因此，对特定于新生儿的 体内 和 体外 研究模型以实现转化相关性的需求很高。由于解剖水平和组织水平之间的相似性、较短的妊娠期和窝产仔数，啮齿动物是研究最多的哺乳动物模型系统。
在这里，我们描述了一种详细的、高度可行的和可重复的程序，用于从 7-9 日龄的小鼠幼崽中分离骨髓及其分化为巨噬细胞的能力。然而，通过使用不同的分化信号，可以实现多种细胞谱系。我们还证明了细胞表面标志物的存在以及骨髓来源的巨噬细胞 （BMDM） 预期的 体外 吞噬活性的存在。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40 &#181;m strainer</td>
			<td>Greiner</td>
			<td>542040</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>96 well round (U) bottom plate</td>
			<td>Thermo Scientific</td>
			<td>12-565-65</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b-BV786</td>
			<td>BD Biosciences</td>
			<td>740861</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse CD206-Alexa Fluor488</td>
			<td>BD Biosciences</td>
			<td>141709</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80-PE</td>
			<td>BD Biosciences</td>
			<td>565410</td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Countess3</td>
			<td>Thermo Scientific</td>
			<td>TSI-C3ACC</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>VWR</td>
			<td>WN182</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Escherichia coli O1:K1:H7</td>
			<td>ATCC</td>
			<td>11775</td>
			<td>Infection</td>
		</tr>
		<tr>
			<td>EVOS FL&#160;</td>
			<td>Invitrogen</td>
			<td>12-563-649</td>
			<td>Cell Imaging System&#160;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Avantor&#160;</td>
			<td>76419-584</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>FluoroBright BMDM</td>
			<td>Thermo fisher Scientific</td>
			<td>A1896701</td>
			<td>Dye free culture media</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Cytiva</td>
			<td>SH30034.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cytiva</td>
			<td>SH30237.01</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>L-929</td>
			<td>ATCC</td>
			<td></td>
			<td>Differentiation</td>
		</tr>
		<tr>
			<td>LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Flowcytometer</td>
		</tr>
		<tr>
			<td>Lysotracker red DND 99</td>
			<td>Invitrogen</td>
			<td>L7528</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>15-010-CV</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin /streptomycin&#160;</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>pHrodo green STP ester&#160;</td>
			<td>Invitrogen</td>
			<td>P35369</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Cell star</td>
			<td>658170</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300120</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Zeiss 710&#160;</td>
			<td>Zeiss</td>
			<td>P20GM103434</td>
			<td>Confocal</td>
		</tr>
	</tbody>
</table>

neonatal mouse bone marrow isolation and preparation of bone marrow-derived macrophages

从成年小鼠中分离骨髓的各种技术已经得到广泛确立。然而，从新生小鼠中分离骨髓具有挑战性且耗时，但对于某些模型来说，它在翻译上是相关和必要的。该方案描述了一种从 7-9 日龄幼崽制备骨髓细胞的有效且直接的方法。然后，这些细胞可以进一步分离或分化为感兴趣的特定细胞类型。巨噬细胞是重要的免疫细胞，在炎症和感染中起主要作用。在发育过程中，新生儿巨噬细胞对组织重塑有显著贡献。此外，新生儿巨噬细胞的表型和功能与成年巨噬细胞不同。该方案还概述了在 L929 条件培养基存在下新生儿巨噬细胞与分离骨髓细胞的分化。使用流式细胞术分析评估分化新生儿巨噬细胞的表面标志物。为了证明功能，还使用 pH 敏感染料偶联的大 肠杆菌测试了吞噬效率。

Bone marrow encloses both hematopoietic and mesenchymal stem cell populations that are self-renewable and can be differentiated into various cell lineages.&#160;Hematopoietic stem cells in the bone marrow give rise to myeloid and lymphoid lineages1. Mesenchymal stem cells produce osteoblasts (bone), adipocytes (fat), or chondrocytes (cartilage)2. These cells have multiple applications in the field of cell biology and tissue engineering, including gene therapy3,4. Progenitor cells present in the bone marrow differentiate into specific cell types in the presence of lineage-specific growth factors. Erythropoietin promotes the proliferation of erythroid progenitor cells, granulocyte colony-stimulating factor (G-CSF) stimulates the growth of neutrophil colonies, and thrombopoietin regulates the production of platelets as a few examples of lineage-specific growth factors5. Cell surface antigen labeled FACS and magnetic-activated cell sorting (MACS) are well-established methods for isolation and purification of the specific bone marrow-derived cell types6.
Though neonatal studies are advancing toward finding the causes of neonatal deaths and addressing the complications during premature births, direct therapeutic development remains an unmet medical need. Smith and Davis stated, &#34;Pediatric patients remain therapeutic orphans&#34;7. There are several challenges, such as small samples, lifelong effects of the outcome, and ethical issues in obtaining consent in clinical studies of neonates8. Hence, there is a high demand for in vivo and in vitro study models specific to neonates to achieve translational relevance. Because of the similarities between anatomical and tissue levels, short gestational periods, and litter sizes, rodents are the most studied mammalian model system.
Here, we describe a detailed, highly feasible, and reproducible procedure for isolating bone marrow from 7-9-day-old mouse pups and their ability to differentiate into macrophages. However, a variety of cell lineages could be achieved with the use of distinct differentiation signals. We also demonstrate the presence of cell surface markers and the presence of in vitro phagocytic activity expected for bone marrow-derived macrophages (BMDMs).

作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

新生小鼠骨髓分离和骨髓来源巨噬细胞的制备

The scope of this study is to understand some of the limitations in the early life immune response to bacterial infection. In order to accomplish this, we need to develop and use research models that are specific to neonates. Isolation of bone marrow from a day-seven-old-pup is challenging and not well established.Neonatal bone marrow derived macrophages are sensitive and prone to stress when compared to other BMDM. The technique used in this protocol is simple and reproducible. This method guides the users in isolating bone marrow from seven to nine-day-old neonatal mice without the use of any enzymes.This study provides a standardized strategy to generate bone marrow-derived macrophages from neonatal mice that can be used to address questions about neonatal immunology. This reproducible method provides an age-specific model for progressing neonatal immune research across many areas of expertise.

使用本研究中概述的方法，可以从 5 只 C57BL/6 小鼠幼崽的窝产仔数中成功分离 25 至 3700 万个骨髓细胞。这种方法已在 5 到 7 只幼崽的窝产仔数中得到验证。在我们的实验中，隔离的最低年龄为 7 天。根据窝产仔数和实验所需的细胞数量少于 100 万，研究人员可以对 7 天以下的小鼠尝试此方案。在作为 M-CSF 来源的 L929 细胞上清液存在下，骨髓细胞在 5 天内分化成巨噬细胞（图 2</stron...

该方案描述了一种非酶和直接的方法，用于分离 7-9 日龄的新生小鼠骨髓细胞，并使用 L929 细胞的上清液作为粒细胞集落刺激因子 （M-CSF） 的来源来产生分化的巨噬细胞。进一步分析骨髓来源的巨噬细胞表面抗原 F4/80 、 CD206 、 CD11b 和功能能力。

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

本研究的范围是了解早期生命对细菌感染的免疫反应的一些局限性。为了实现这一目标，我们需要开发和使用特定于新生儿的研究模型。从 7 日龄幼犬中分离骨髓具有挑战性且尚未得到充分证实。与其他 BMDM 相比，新生儿骨髓来源的巨噬细胞敏感且容易受到压力。该协议中使用的技术简单且可重复。该方法指导用户在不使用任何酶的情况下从 7 至 9 日龄的新生小鼠中分离骨髓。本研究提供了一种从新生小鼠生成骨髓来源的巨噬细胞的标准化策略，可用于解决有关新生儿免疫学的问题。这种可重复的方法为在许多专业领域推进新生儿免疫研究提供了一种针对特定年龄的模型。

neonatal-mouse-bone-marrow-isolation-preparation-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

995 次观看.  West Virginia University School of Medicine. 本研究的范围是了解早期生命对细菌感染的免疫反应的一些局限性。为了实现这一目标，我们需要开发和使用特定于新生儿的研究模型。从 7 日龄幼犬中分离骨髓具有挑战性且尚未得到充分证实。与其他 BMDM 相比，新生儿骨髓来源的巨噬细胞敏感且容易受到压力。该协议中使用的技术简单且可重复。该方法指导用户在不使用任何酶的情况下从 7 至 9 日龄的新生小鼠中分?...

视频: 作者聚焦：开发一种从新生小鼠中分离骨髓来源的巨噬细胞的可重复方法，以促进早期免疫反应

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

科研

免疫与感染

Non-Enzymatic Method for Isolating Neonatal Mouse Bone Marrow Cells

After decapitation, sterilize the body of the anesthetized neonate mouse with 70%ethanol. Using forceps and fine tipped surgical scissors, make an incision between the abdomen and hind legs. Then remove the skin by pulling it toward the foot of the hind limbs.Scrape the connective tissue and muscle attached to the bones using sharp edged forceps. Cut the tibia and femur with scissors. Use forceps to place the tibia and femur in a MEM culture media tube on ice.Transfer the bones to a 40 micron strainer with a collection tube. Crush the bones with a three milliliter syringe plunger against the strainer to release the marrow. Centrifuge the cell suspension at 350 G for five minutes at four degrees Celsius.Aspirate the supernatant and gently re-suspend the cells in 0.2%sodium chloride to make a hypotonic solution for the lysis of erythrocytes. Immediately add an equal volume of 1.6%sodium chloride to make the solution isotonic. Centrifuge the supernatant and remove the lysis solution.Re-suspend the cells in complete DMEM and count the cells on an automated cell counter.

分离新生小鼠骨髓细胞的非酶方法

Differentiation of Neonatal Bone Marrow-Derived Macrophages Using a Supernatant of L929 Cells

To begin, seed 20 million neonate mouse bone marrow cells per T75 flask in 10 milliliters of complete DMEM containing 2.5 milliliters of 10%L929 cell supernatant. Incubate the culture flask at 37 degrees Celsius with 5%carbon dioxide for five days. On day three of differentiation, add two milliliters of L929 conditioned media into the flask.Under an inverted microscope with 20x magnification, daily observe the cells. To harvest the macrophages on day five, aspirate the differentiation media from the flask. Wash the cells with five milliliters of PBS to remove non-adherent cells and serum proteins.Add five milliliters of 0.05%trypsin EDTA to the flask and incubate at 37 degrees Celsius incubator with 5%carbon dioxide. After five minutes, add five milliliters of DMEM and pipette to detach the cells. Centrifuge the cell suspension at 350 g for five minutes.Dissociate the cell palate in one milliliter of complete DMEM. Count the cells on an automated cell counter. In the presence of L929 cell supernatant, bone marrow cells were differentiated into macrophages within five days.Spindle-shaped cell formation was observed from the second day with nearly half displaying this morphology by day three and a majority by day five.

使用 L929 细胞上清液分化新生儿骨髓来源的巨噬细胞

Assessing Phagocytic Function of Neonatal Bone Marrow-Derived Macrophages Using a Fluorescent Bacterial Uptake Assay

To begin resuspend the mouse neonatal bone marrow derived macrophages in complete DMEM without phenol red or antibiotics. Seed 500 microliters of cell suspension at a density of 200, 000 per quadrant in a 35 millimeter quad dish. Remove the precalculated stock of Escherichia coli from minus 80 degrees Celsius.Aliquot the desired volume of cell suspension into a 1.7 milliliter micro centrifuge tube to prepare bacterial inoculum at a multiplicity of infection of 25, then add PBS to a total volume of one milliliter. Centrifuge the bacterial suspension at 2000 G for five minutes and resuspend the pellet in 50 microliters of PBS. Add green fluorescent pH sensitive dye to a final concentration of 500 micro molars and incubate for 20 minutes in the dark for dye conjugation.Wash the bacteria four times with one milliliter of PBS by centrifugation at 1000 G for five minutes. Resuspend the pellet in 500 microliters of complete DMEM without phenol red and add the bone marrow derived macrophage cultures. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for four hours.Add 200 nanograms of cell permeable red fluorescent dye to stain the lysosomes, Upon bacterial infection, abundant green fluorescent bacteria phagocytosed inside bone marrow derived macrophages were detected. The green fluorescence further localizes with red fluorescence indicative of acidified lysosomes. The phagocytosis and appearance of green pH sensitive dipositive neonatal bone marrow derived macrophages were also observed throughout the four hour infection.