Name: Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice
Uploaded: 2023-10-06T13:40:00
Duration: 1 min 32 s
Description: Watch this Scientific Journal Video about Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice at JoVE.com

isolation of cns cell types using magnetic beads in naive and eae mice

从自身免疫小鼠中同时分离所有 CNS 驻留细胞类型

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage, gliosis, and neurodegeneration. Despite numerous research approaches in this field, the pathophysiology of MS is still not fully understood1,2,3,4. The most common animal model for investigating MS is the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) which shares many of its clinical and pathophysiological features5,6,7,8,9. It is based on the response of the immune system against CNS-specific antigens leading to inflammation, demyelination, and neuroaxonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is a suitable model for the investigation of neuroinflammatory pathways and signaling cascades found in MS.
Current therapy options for MS are only partially effective and focus primarily on the initial inflammatory phase of the disease. However, the neurodegenerative component of MS seems to be the major challenge for long-term therapeutic approaches. Therefore, reproducible, and precise cell isolation protocols are required to investigate molecular and cellular mechanisms in autoimmune diseases in a comprehensive way. Even if some protocols for the isolation of one single cell type exist10,11,12,13,14,15, there is an unmet need for the simultaneous isolation of several CNS-resident cell populations at once. Previous protocols for the isolation of CNS-resident cells lack in preserving cellular functionality and purity, resulting in co-cultivation with neighboring cells16,17,18 or the unsuitability for complex analyses of intracellular networks ex vivo19,20,21,22.
The aim of this protocol was to establish a reproducible and comprehensive method for the simultaneous isolation of pure viable single-cell suspensions of all principal CNS-resident cell types applicable in adult healthy and EAE mice. The different cell types were isolated using magnetic-activated cell sorting (MACS)23. The cell separation can be accomplished either by positive selection, i.e., magnetic labeling of cell-type-specific surface markers, or by negative selection via biotinylation and depletion of all undesired cells. Flow cytometry was applied to ensure a purity of above 90% and a viability of at least 80% of the isolated single-cell suspensions.
In conclusion, the main goal was to establish a protocol for the simultaneous isolation of all main CNS-resident cell types as a versatile tool for the investigation of neuroinflammatory pathways offering a comprehensive and precise analysis of complex cellular networks and biochemical signaling cascades in healthy and EAE mice.

作者聚焦：通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and new inflammatory pathways, ex vivo from one single cell suspension, applicable to healthy mice and those with experimental autoimmune encephalomyelitis.Additionally, mice numbers are decreased Our protocol for the simultaneous isolation of all four major CNS-resident cell types in healthy and EAE mice could be used as a helpful tool for research groups studying new inflammatory pathways, allowing a much more accurate analysis of complex biomolecular mechanisms and cellular networks ex vivo. New scientific questions that could be answered with the help of our protocol are dynamic investigations in EAE during different stages of disease course, like neuroinflammation, neurodegeneration, and remission. Also, cell-cell interactions and biochemical pathways could be studied on an individual level.Likewise, functional assays could be performed with cultivated cells. We intend to focus on dynamic studies of the EAE course for multi-omic analysis from one individual CNS homogenate per EAE time point.

Watch this Scientific Journal Video about Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice at JoVE.com

Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice  

在幼稚小鼠和EAE小鼠中使用磁珠分离CNS细胞类型

首先，将纯化的未稀释小鼠CNS细胞悬液分成两部分，以进一步分离小胶质细胞和少突胶质细胞。然后将针对不同中枢神经系统细胞类型的表面抗原特异性的磁性标记微珠添加到相应的细胞悬液中。将色谱柱置于磁场中，使色谱柱相等，然后将得到的细胞悬浮液添加到色谱柱上。磁性标记的细胞将保留在色谱柱内，未标记的细胞将贯穿其中。从磁场中取出色谱柱后，将磁性标记的细胞从色谱柱中冲洗到试管中，作为阳性选择的细胞部分。将来自少突胶质细胞的负流分成两部分，以进一步分离神经元和星形胶质细胞。使用细胞类型特异性标记物结合活细胞或死细胞鉴别的流式细胞术分析产生了小胶质细胞、少突胶质细胞、星形胶质细胞和神经元。不同细胞群的表型表征证明，所有主要的中枢神经系统驻留细胞类型均获得了纯度和活力约为 90% 的单细胞悬液。

Research

JoVE Journal

Neuroscience

Isolation of CNS Cell Types Using Magnetic Beads in Naive and EAE Mice

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...