小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽
Summary
Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.
Abstract
Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique – described here – is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer’s or Parkinson’s disease.
Introduction
人类大脑包含估计为85十亿神经元和另一个85十亿非神经元细胞，包括神经胶质1。对于过去的100年神经科学家主要集中在神经元细胞群的大部分时间里，相信神经胶质细胞比被动的支持细胞为神经元提供结构支持更小 – “神经胶质细胞”翻译成英语的，因此希腊词源“胶水”。然而，最近它已成为日益明显的是，神经细胞 – 胶质细胞的相互作用可能是更为基本的神经生物学，神经生理学和许多神经变性疾病的发生和进展的基本方面。小脑颗粒细胞（信用担保公司），最丰富的神经元的同质人口在人的大脑，支配小脑和弥补90％以上，其细胞成分。因此，这些细胞已被广泛用于在体外作为吨的模型系统他研究神经发育，功能和病理2-6。
不过，CGC文化还含有小胶质细胞和神经胶质细胞等在争议显著的比例。其结果是，CGC数据推定显示直接神经元对不同的细胞治疗可能实际上出现 – 在部分或全部 – 从在培养相邻神经胶质的间接二次响应。为了评估这一点，我们选择的除去从CGC神经元培养小胶质用的L-亮氨酸甲酯（LME）的助剂。 LME是一个lysomotropic剂原本用于选择性地破坏巨噬细胞7，并一直使用也选择性地从神经元，星形胶质细胞和神经胶质混合培养8,9,10消耗小胶质细胞。 LME由巨噬细胞和小胶质细胞内化，其中它引起溶酶体破裂和随后的细胞凋亡13,14。巨噬细胞和小胶质细胞是典型丰富的溶酶体，使他们成为particula暴露于LME治疗RLY脆弱。该协议提供了一个功能强大，简单易用的方式，以确定小胶质细胞在利用CGC和其他神经元/胶质细胞培养系统实验的贡献。
Protocol
Representative Results
Discussion
最重要的步骤，以确保成功选择性消除小胶质细胞来自CGC和/或混合培养物有：1）保持无菌，健康的文化CGC; 2）过滤灭菌的含LME介质并返回该溶液至pH 7.4; 3）保持保留CGC媒体和LME-含37媒体 °C，这样可以避免热冲击;和4）迅速工作，以减少单元保持在培养箱外的时间。
我们使用25毫米LME从我们的CGC文化耗尽胶质 – 以前在我们的实验室进行了优化的浓度。尽管如此，一些?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.
Materials
| Forceps | Sigma-Aldrich | F4142 | The curved end facilitates removal of the cerebellum |
| Micro-dissecting scissors | Sigma-Aldrich | S3146 | Straight, sharp point facilitates rodent P4-7 dissection |
| L-leucine methyl ester hydrochloride | Sigma-Aldrich | 7517-19-3 | |
| EBSS solution | Sigma-Aldrich | E7510-500 ml | |
| Poly-D-lysine | Sigma-Aldrich | 27964-99-4 | Coat coverslips 1 day before use |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A9418 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich | P4417 | |
| DNase | Sigma-Aldrich | D5025 | |
| Soybean trypsin inhibitor | Sigma-Aldrich | T6414 | |
| Mouse anti-ED1 antibody | Abcam | ab31630 |
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