Flow Cytometric Analysis and Sorting of Enteric Nervous System Cells: From Human to Mouse, an Optimized Protocol

Isolation of neurons and glial cells from the enteric nervous system (ENS) enables ex-vivo studies, including 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 compared different protocols to optimize tissue dissociation of mouse colons. A 30-minute 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 (RT-qPCR) on sorted fractions. Overall, the mouse ENS expression profile significantly overlapped with the human one, confirming that current dissociation protocols yield a mixed staining pattern 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, at the TIP of the CD56/CD24 population. In conclusion, we present here a protocol for high purity FACS-based isolation of viable enteric neurons and glial cells, suitable for downstream applications.