Isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation

Charles J. Benck, Tijana Martinov, Brian T Fife, Devavani Chatterjea

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

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.

Original languageEnglish (US)
Article numbere53638
JournalJournal of Visualized Experiments
Volume2016
Issue number107
DOIs
StatePublished - Jan 25 2016

Keywords

  • Collagenase digestion
  • Density gradient centrifugation
  • Flow cytometry
  • Immunology
  • Issue 107
  • Lymphocytes
  • Skin
  • T cells
  • Tissue dissociation

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