从成年小鼠脊髓中选择性高效分离灰质星形胶质细胞的方法。

IF 3.3 3区 医学 Q2 NEUROSCIENCES
Ryoma Iwasaki, Yuta Kohro, Makoto Tsuda
{"title":"从成年小鼠脊髓中选择性高效分离灰质星形胶质细胞的方法。","authors":"Ryoma Iwasaki, Yuta Kohro, Makoto Tsuda","doi":"10.1186/s13041-024-01097-3","DOIUrl":null,"url":null,"abstract":"<p><p>A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2<sup>+</sup> cells were divided into two distinct populations, ACSA-2<sup>high</sup> and ACSA-2<sup>low</sup>. By analyzing the expression of cell-type marker genes, the ACSA-2<sup>high</sup> and ACSA-2<sup>low</sup> populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2<sup>high</sup> cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.</p>","PeriodicalId":18851,"journal":{"name":"Molecular Brain","volume":null,"pages":null},"PeriodicalIF":3.3000,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11106874/pdf/","citationCount":"0","resultStr":"{\"title\":\"A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice.\",\"authors\":\"Ryoma Iwasaki, Yuta Kohro, Makoto Tsuda\",\"doi\":\"10.1186/s13041-024-01097-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2<sup>+</sup> cells were divided into two distinct populations, ACSA-2<sup>high</sup> and ACSA-2<sup>low</sup>. By analyzing the expression of cell-type marker genes, the ACSA-2<sup>high</sup> and ACSA-2<sup>low</sup> populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2<sup>high</sup> cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.</p>\",\"PeriodicalId\":18851,\"journal\":{\"name\":\"Molecular Brain\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-05-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11106874/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Brain\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1186/s13041-024-01097-3\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"NEUROSCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Brain","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s13041-024-01097-3","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
引用次数: 0

摘要

越来越多的证据表明,大脑中的星形胶质细胞具有区域内和区域间的异质性。然而,由于缺乏从脊髓分离星形胶质细胞的有效方法,人们对成年小鼠脊髓星形胶质细胞的异质性知之甚少。在这项研究中,我们开发了一种新方法,利用地衣芽孢杆菌的冷活性蛋白酶和星形胶质细胞表面抗原-2(ACSA-2)抗体从成年小鼠体内分离脊髓星形胶质细胞。利用荧光激活细胞分拣技术,将分离出的脊髓 ACSA-2+ 细胞分为两个不同的群体,即 ACSA-2high 和 ACSA-2low。通过分析细胞类型标记基因的表达,ACSA-2高细胞群和ACSA-2低细胞群分别被鉴定为星形胶质细胞和上皮细胞。此外,ACSA-2高细胞的mRNA编码基因在灰质(GM)中大量表达,而在白质星形胶质细胞中却没有表达。通过优化酶分离程序,GM 星形胶质细胞的产量也有所增加。因此,我们新建立的方法能从成年小鼠的脊髓中选择性地、高效地分离出GM星形胶质细胞,可用于生理和病理条件下的批量或单细胞RNA测序。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice.

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Molecular Brain
Molecular Brain NEUROSCIENCES-
CiteScore
7.30
自引率
0.00%
发文量
97
审稿时长
>12 weeks
期刊介绍: Molecular Brain is an open access, peer-reviewed journal that considers manuscripts on all aspects of studies on the nervous system at the molecular, cellular, and systems level providing a forum for scientists to communicate their findings. Molecular brain research is a rapidly expanding research field in which integrative approaches at the genetic, molecular, cellular and synaptic levels yield key information about the physiological and pathological brain. These studies involve the use of a wide range of modern techniques in molecular biology, genomics, proteomics, imaging and electrophysiology.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信