Protocol for isolating immune cells from human gastric muscularis propria for single-cell analysis

Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45⁺ cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al.¹.