Flow Cytometry and Single-Cell Analysis for Characterizing Microglia Activation in Early Postnatal Mouse Brain...

Microglia, the brain's resident immune cells, exhibit region and context-specific transcriptional profiles during development and disease. This protocol presents two complementary methods for studying microglial populations in mouse cerebellar tissue: flow cytometry and single-cell RNA sequencing. As the first method used, the flow cytometry-based protocol is optimized for early postnatal brains, ensuring robust cell isolation and consistency across experimental conditions. It begins with tissue dissociation using enzymatic digestion, followed by myelin removal via Percoll density gradient centrifugation to yield a high-quality neural cell suspension, and a gating strategy based on CD45 and CD11b expression. Live/dead staining ensures cell viability, and fluorochrome-conjugated antibodies are used to profile the expression of selected surface markers on microglia. Compensating controls are performed using latex beads with validated gating strategies using fluorescence minus one (FMO) control. The second method involves single-cell RNA sequencing using 10X Genomics, following the same upstream isolation steps, which enable transcriptomic profiling of microglia across conditions. Microglial clusters are identified using gene expression analysis, and differential expression analysis is conducted between experimental groups. A random forest classifier is applied solely to distinguish male and female samples when multiplexed. Together, these reproducible and adaptable protocols provide a robust framework for investigating microglial diversity in the cerebellum during early brain development and its potential alteration following experimental perturbations.