Isolation and Characterization of Immune Cells from Mouse Skin in Steady State and During Inflammation

Abstract

Abstract Immune cells in the skin play an important role in maintaining tissue homeostasis and responding to tissue stress and injury. However, isolating immune cells from skin can be challenging because of its dense extracellular matrix and the potential effects of prolonged enzymatic digestion on cell viability and surface markers. Here, we describe a combined mechanical and enzymatic method for isolating immune cells from mouse skin for downstream flow cytometry and RNA sequencing applications, together with a flow cytometry gating strategy for the identification and characterization of skin myeloid cell populations. The long digestion uses an overnight dispase II treatment followed by collagenase IV and DNase I digestion to facilitate epidermal and dermal separation and subsequent tissue dissociation, while the fast‐digest bypasses the overnight step and uses Liberase TL to obtain cells within approximately 2 hr. The fast‐digest workflow reduces processing time and provides an alternative approach for applications requiring RNA‐based analyses. Together, these workflows provide flexible methods for studying immune cell composition and transcriptional responses during steady‐state and injury. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol : Isolation of skin immune cells for flow cytometry Alternative Protocol : Isolating skin immune cells using a “fast‐digest” technique

The authors' abstract, as published at the source. Current Protocols, 2026 · DOI ↗