Protocol for the isolation of human buccal cells for single-cell applications

Human buccal mucosa contains epithelial, stromal, and resident immune cell populations, but isolating these cells as a high-viability single-cell suspension is technically challenging. Here, we describe a gentle dissociation workflow for human buccal punch biopsies that combines controlled enzymatic digestion (collagenase and nuclease) with optimized speed, temperature, and incubation time to efficiently release heterogeneous cell populations while minimizing cell damage. The protocol reliably yields a heterogeneous single-cell suspension compatible with flow cytometry and single-cell RNA sequencing, with typical viabilities ≥80%.