Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Cindy Bokobza; Alice Jacquens; Manuela Zinni; Val&#233;rie Faivre; Jennifer Hua; David Guenoun; Caroline Userovici; Shyamala Mani; Vincent Degos; Pierre Gressens; Juliette Van Steenwinckel

doi:10.3791/62964

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

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07:23 min

July 25, 2022

DOI: 10.3791/62964-v

Cindy Bokobza*¹, Alice Jacquens*^1,2, Manuela Zinni¹, Valérie Faivre¹, Jennifer Hua¹, David Guenoun¹, Caroline Userovici¹, Shyamala Mani¹, Vincent Degos^1,2, Pierre Gressens¹, Juliette Van Steenwinckel¹

¹NeuroDiderot, Inserm UMR-1141, Hôpital Robert Debré 48,Université de Paris, ²Department of anesthesia and critical care,APHP-Sorbonne university

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Overview

This study presents a reproducible protocol for isolating primary microglia from neonatal mice, using magnetic sorting technology. The methodology allows the culture of microglia under conditions that closely replicate in vivo characteristics, providing insights into their phagocytic activity.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Microglia Research

Background

Microglia play crucial roles in the central nervous system.
Understanding microglial responses to stimuli is important for neuroinflammatory research.
Magnetic cell sorting provides a viable strategy for isolating microglia.
Previous protocols may lack reproducibility or relevance to in vivo conditions.

Purpose of Study

To develop a protocol for efficiently isolating and culturing primary microglia.
To allow for the assessment of microglial responses to inflammatory stimuli.
To evaluate the purity and viability of isolated microglia.

Methods Used

Cell culture method utilizing magnetic sorting to isolate microglia from neonatal mice.
The biological model involves cortical microglia from neonate pups.
Immunochemistry and flow cytometry were employed to assess cell purity.
Key steps include dissection, enzymatic dissociation, and sorting using CD11b microbeads.
Phagocytic assays evaluated microglial activity in response to pro-inflammatory factors.

Main Results

Microglia demonstrated increased phagocytic activity in response to specific inflammatory conditions.
Increased cell viability and purity were confirmed through flow cytometry post-sort.
Confocal microscopy highlighted pronounced differences in phagocytic activity based on stimulation duration.
The method clarified distinctions between different brain cell populations.

Conclusions

This study establishes a reliable method for isolating mouse microglia for in vitro experimentation.
The findings enhance the understanding of microglial functions and their responses to inflammation.
This protocol serves as a foundation for further investigations into neuroinflammatory processes.

Frequently Asked Questions

What are the advantages of using this isolation method for microglia?

This method ensures high purity and viability of isolated microglia, closely mimicking in vivo conditions, which enhances experimental relevance.

How are microglia cultured after isolation?

After isolation, microglia are cultured in specialized medium and stimulated with inflammatory factors to assess their functional responses.

What outcomes can be measured using this protocol?

Outcomes include microglial viability, phagocytic activity, and response to inflammatory stimuli assessed via confocal microscopy and flow cytometry.

Can this method be adapted for other types of brain cells?

While this protocol is specifically designed for microglia, adaptations may be possible for other cell types by modifying the sorting antibodies.

What are the limitations of this microglia isolation protocol?

Limitations may include the need for precise dissection and careful handling to avoid mechanical damage to cells during sorting.

What type of analysis can be performed after isolating microglia?

Following isolation, various analyses such as transcriptomic studies, immunochemistry, and functional assays can be conducted to explore microglial roles.

原代小胶质细胞培养通常用于评估新的抗炎分子。本协议描述了一种可重复且相关的方法，用于从新生儿幼崽中磁性分离小胶质细胞。

该协议使我们能够在与体内特征非常相似的条件下培养小胶质细胞。使用磁性细胞分选技术，我们的方案允许我们在体外仅两天刺激小胶质细胞，而无需在培养基中使用任何血清。首先，用小剪刀从颈部到鼻子剪开皮肤，沿着 15 到 20 毫米的矢状缝合线。

插入尖端，大孔与颅骨平行。从两侧切到眼睛。用小剪刀剪开眼睛之间，将头骨和大脑与头部分离。

用两个镊子抓住靠近嗅球的头骨，然后小心地撕裂头骨。用剃须刀片去除小脑和嗅觉球，将大脑切成两块。将脑碎片放入含有40毫升不含钙和镁的HBSS的培养皿中。

根据此表制备解离混合物。将 12 个脑碎片转移到 C 管中，每个解离管的总重量为 1.2 克。然后将C管放在解离器上加热。

在解离器中启动优化的NTDK程序。离心20秒。通过移液三次完成机械解离。

将细胞转移到四个带有过滤器的15毫升管中。用10毫升含有钙和镁的HBSS冲洗过滤器。离心10分钟，用10毫升移液管除去上清液。

小心地加入10毫升含有钙和镁的HBSS，然后重新悬浮沉淀。再次，离心并除去上清液。用六毫升分选缓冲液重新悬浮沉淀。

重复离心并弃去上清液。然后加入 200 微升 CD11b 微珠溶液，并将试管在 4 摄氏度下孵育 15 至 20 分钟。孵育后，用六毫升分选缓冲液重新悬浮沉淀。

重复离心并用八毫升分选缓冲液重新悬浮沉淀。接下来，按照分离器上的波塞尔程序准备八列。通过一次添加一毫升细胞悬液使细胞通过色谱柱。

用一毫升分选缓冲液，在无菌洗脱板上洗脱CD11b阳性细胞。将细胞汇集在50毫升管中。离心并用10毫升冷小胶质细胞培养基重新悬浮沉淀。

计数CD11b阳性细胞。将细胞重新悬浮在冷小胶质细胞培养基中，以获得每毫升650，000至700，000个细胞的最终浓度。将悬浮液分配到细胞培养板中。

将板在 37 摄氏度下用 5% 二氧化碳孵育过夜。第二天，用预热的小胶质细胞培养基替换培养基，并重复孵育过夜。在此步骤之前刺激细胞，并在刺激的最后三个小时内进行吞噬测定。

根据此表以每个细胞50个珠子的比例计算磁珠数量。准备珠子混合物。将试管在 37 摄氏度的水浴中孵育一小时。

每10分钟涡旋一次。向每个孔中加入计算出的珠状溶液体积并孵育三小时。使用这种方法，在通过促炎因子（例如白细胞介素-1 β）加干扰素 γ 或脂多糖刺激后评估小胶质细胞的吞噬活性。

刺激6至24小时后，用共聚焦显微镜分析荧光Cy3小胶质细胞珠。6小时后，小胶质细胞仅在白细胞介素-1 β加干扰素γ条件下开始吞噬Cy3磁珠。24小时后，两种刺激的Cy3荧光都有所增加，突出了吞噬活性的增加。

流式细胞术提高了小胶质细胞培养物的纯度，分选后细胞活力增加。使用流式细胞术和RT-qPCR细胞群标记物分析出生后第八天从小鼠大脑中分选CD11b阳性细胞之前和之后。这些分析区分了各种脑细胞群，例如用于小胶质细胞的CX3CR135，用于少突胶质细胞的O4或OLIG2，用于神经元的NeuN或突触素，以及用于星形胶质细胞的ACSA-2或GFAP。

使用CD11b抗体进行细胞分选后，仅获得小胶质细胞。重要的是在将悬浮液添加到色谱柱时非常轻柔地移液，以防止堵塞并避免机械细胞死亡。在这种方法之后，可以进行转录组蛋白质组学，例如蛋白质血液或Luminex，甚至免疫化学，以查看小胶质细胞刺激的效果。

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