Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity
Summary: Numerous approaches have been developed to isolate microglia from the brain, but procedures using enzymatic dissociation at 37°C can introduce drastic transcriptomic changes and confound results from gene expression assays. Here, we present an optimized protocol for microglia isolation usin...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722005500 |
| _version_ | 1811292682383785984 |
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| author | Shawn Herron Jean-Christophe Delpech Charlotte Madore Tsuneya Ikezu |
| author_facet | Shawn Herron Jean-Christophe Delpech Charlotte Madore Tsuneya Ikezu |
| author_sort | Shawn Herron |
| collection | DOAJ |
| description | Summary: Numerous approaches have been developed to isolate microglia from the brain, but procedures using enzymatic dissociation at 37°C can introduce drastic transcriptomic changes and confound results from gene expression assays. Here, we present an optimized protocol for microglia isolation using mechanical homogenization. We use Dounce homogenization to homogenize mouse brain tissue into single-cell suspension. We then isolate microglia through Percoll gradient and flow cytometry. Isolated microglia exhibit a gene expression pattern without the changes induced by heated enzymatic digestion.For complete details on the use and execution of this protocol, please refer to Clayton et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-13T04:49:03Z |
| format | Article |
| id | doaj.art-adaec674aa724706a9101d53135bbef5 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-13T04:49:03Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-adaec674aa724706a9101d53135bbef52022-12-22T03:01:45ZengElsevierSTAR Protocols2666-16672022-12-0134101670Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrityShawn Herron0Jean-Christophe Delpech1Charlotte Madore2Tsuneya Ikezu3Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA; Corresponding authorLaboratoire NutriNeuro, UMR 1286, Bordeaux INP, INRAE, University of Bordeaux, Bordeaux, FranceLaboratoire NutriNeuro, UMR 1286, Bordeaux INP, INRAE, University of Bordeaux, Bordeaux, FranceDepartment of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA; Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL, USA; Corresponding authorSummary: Numerous approaches have been developed to isolate microglia from the brain, but procedures using enzymatic dissociation at 37°C can introduce drastic transcriptomic changes and confound results from gene expression assays. Here, we present an optimized protocol for microglia isolation using mechanical homogenization. We use Dounce homogenization to homogenize mouse brain tissue into single-cell suspension. We then isolate microglia through Percoll gradient and flow cytometry. Isolated microglia exhibit a gene expression pattern without the changes induced by heated enzymatic digestion.For complete details on the use and execution of this protocol, please refer to Clayton et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722005500Classification Description: Cell biologyCell isolationFlow cytometry/Mass cytometryCell separation/fractionationRNAseqMolecular biology |
| spellingShingle | Shawn Herron Jean-Christophe Delpech Charlotte Madore Tsuneya Ikezu Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity STAR Protocols Classification Description: Cell biology Cell isolation Flow cytometry/Mass cytometry Cell separation/fractionation RNAseq Molecular biology |
| title | Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| title_full | Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| title_fullStr | Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| title_full_unstemmed | Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| title_short | Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| title_sort | using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity |
| topic | Classification Description: Cell biology Cell isolation Flow cytometry/Mass cytometry Cell separation/fractionation RNAseq Molecular biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722005500 |
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