Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing

Pericytes, as a core component of the neurovascular unit, play a vital role in maintaining the integrity of the blood-brain barrier and regulating cerebral hemodynamic homeostasis through modulation of microvascular tone. Dysfunction of pericytes has been implicated in a variety of neurological disorders. To investigate the heterogeneity of pericytes and elucidate their mechanisms in neurovascular diseases at single-cell resolution, we established an optimized and reproducible protocol for isolating mouse brain pericytes suitable for single-cell RNA sequencing (scRNA-seq). This method builds upon the established CD13⁺/CD31^- sorting strategy, but incorporates key enhancements tailored specifically for scRNA-seq applications. Briefly, brains were harvested from three adult C57BL/6 mice. The brain tissues were mechanically dissociated and subjected to enzymatic digestion to obtain single-cell suspensions. After sequential centrifugation, the final cell pellet was resuspended in 20% bovine serum albumin (BSA) solution, a pivotal step we introduced to minimize stress and aggregation, thereby maximizing viability and RNA quality for sequencing. This optimized FACS-based approach thus enables the robust isolation of viable mouse brain pericytes meeting the stringent requirements for scRNA-seq, offering a powerful tool for dissecting pericyte heterogeneity and their pathological roles in neurovascular diseases.