Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity

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). [Display omitted] •Optimized protocol for mouse microglia isolation without an enzymatic dissociation step•Using Dounce homogenization at 4°C to generate single-cell suspension•Isolation of microglia through Percoll gradient and flow cytometry•Can preserve transcriptomic integrity and is suitable for multiple sequencing endpoints Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.