Mechanical isolation of neonatal and adult mouse dura leukocytes for flow cytometry analysis

• Published in: STAR protocols 2023-06, Vol.4 (2), p.102272-102272, Article 102272
• Main Authors: Niu, Chunxiao, Jiang, Hui, Wu, Mengyao, Zou, Tao, Wu, Xian, Lu, Yuchen, Deng, Lijiao, Guo, Tingting, Si, Chuan-Yimu, Zhang, Jiyan
• Format: Article
• Language: English
• Subjects: Cell Biology; Immunology; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102272

The meninges, consisting of the pia, arachnoid, and dura layers, provide immunosurveillance of the central nervous system with both innate and adaptive immune cells. Here we present an optimized protocol for isolating dura leukocytes from neonatal and adult mice. We describe steps for harvesting the skull cap, extracting the dura mater, mechanical isolation of dura leukocytes, and flow cytometry analysis. Unlike the time-consuming enzymatic digestion isolation which makes dura hematopoietic stem cells (HSCs) undetectable, this rapid and simplified technique permits dura HSC identification. For complete details on the use and execution of this protocol, please refer to Niu et al. (2022).1 [Display omitted] •Optimized protocol for mechanical isolation of dura leukocytes•Rapid and simplified technique to obtain dura leukocytes•Suitable for flow cytometry analysis of HSCs and downstream progenitors•Suitable for single-cell RNA sequencing or colony-forming unit cell assay Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The meninges, consisting of the pia, arachnoid, and dura layers, provide immunosurveillance of the central nervous system with both innate and adaptive immune cells. Here we present an optimized protocol for isolating dura leukocytes from neonatal and adult mice. We describe steps for harvesting the skull cap, extracting the dura mater, mechanical isolation of dura leukocytes, and flow cytometry analysis. Unlike the time-consuming enzymatic digestion isolation which makes dura HSCs undetectable, this rapid and simplified technique permits dura HSC identification.