Flow Cytometric Analysis and Sorting of Murine Enteric Nervous System Cells: An Optimized Protocol

Isolation of neurons and glia from the enteric nervous system (ENS) enables ex vivo studies, including the analysis of genomic and transcriptomic profiles. While we previously reported a fluorescence-activated cell sorting (FACS)-based isolation protocol for human ENS cells, no equivalent exists for mice. As directly applying the human protocol to mouse tissue resulted in low recovery of live ENS cells, we optimized tissue dissociation using mouse colons. A 30 min Liberase-based digestion showed optimal recovery of viable ENS cells, with CD56 and CD24 emerging as the most reliable markers to select and subdivide these cells. ENS' identity was further validated by FACS, using neuronal (TUBB3) and glial (SOX10) markers and reverse transcriptase quantitative PCR on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, showing that current dissociation protocols yield a mixed population of enteric neurons and glia. Nonetheless, using the imaging flow cytometer BD S8 FACS Discover and ELAVL4 as a neuronal soma-associated marker, we observed enrichment of neurons in a CD56/CD24^TIP population. In conclusion, we present here a protocol for high-purity FACS-based isolation of viable mouse ENS cells, suitable for downstream applications.