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Updated: Mar 26, 2026
Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation
Published on: January 25, 2016
Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation
Charles J Benck1, Tijana Martinov2, Brian T Fife2
1Department of Biology, Macalester College.
Insights
This study presents an economical method for dissociating mouse skin into single cells using collagenase D and density gradients. The protocol yields viable immune cells suitable for flow cytometry and other analyses.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Single-cell suspension of murine skin is crucial for analyzing immune cells in skin inflammation models.
- Existing methods for skin dissociation can be costly or compromise cell integrity and surface protein expression.
Purpose of the Study:
- To describe an effective and economical protocol for dissociating mouse skin into single-cell suspensions.
- To provide a method that preserves cell viability and surface marker integrity for downstream analyses.
Main Methods:
- Digestion of mouse skin using collagenase D in a nutrient-rich solution.
- Separation of hematopoietic cells via discontinuous density gradient centrifugation.
- Analysis of obtained cells using flow cytometry.
Main Results:
- The protocol yielded highly viable single-cell suspensions from murine skin.
- Immune cell subsets were successfully isolated and characterized.
- No adverse effects on common surface marker proteins used in flow cytometry were observed.
Conclusions:
- This collagenase D-based protocol offers an economical and efficient alternative for preparing murine skin-derived single cells.
- The method is suitable for various downstream applications, including in vitro studies, in vivo transfer, and flow cytometry.
- The protocol preserves cellular viability and surface marker integrity, ensuring reliable phenotypic and functional characterization of immune cells.
Abstract:
Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.
