Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing

Sebastian Willenborg; Juliana G Roscito; Alexander Gerbaulet; Axel Roers; Andreas Dahl; Sabine A Eming; Susanne Reinhardt

doi:10.1016/j.xpro.2022.101337

Isolation of macrophages from mouse skin wounds for single-cell RNA sequencing

STAR Protoc. 2022 Apr 19;3(2):101337. doi: 10.1016/j.xpro.2022.101337. eCollection 2022 Jun 17.

Authors

Sebastian Willenborg¹, Juliana G Roscito², Alexander Gerbaulet³, Axel Roers³, Andreas Dahl², Sabine A Eming^{1

4

5

6}, Susanne Reinhardt²

Affiliations

¹ Department of Dermatology, University of Cologne, 50937 Cologne, Germany.
² DRESDEN-concept Genome Center (DcGC), Center for Molecular and Cellular Bioengineering, TU Dresden, 01307 Dresden, Germany.
³ Institute for Immunology, Medical Faculty, TU Dresden, 01307 Dresden, Germany.
⁴ Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany.
⁵ Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50674 Cologne, Germany.
⁶ Institute of Zoology, Developmental Biology Unit, University of Cologne, 50674 Cologne, Germany.

Abstract

Understanding macrophage heterogeneity in tissue repair is a major challenge. Here, we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing. We use a modified version of the original Smart-seq2 protocol to increase speed and accuracy. This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation. For complete details on the use and execution of this protocol, please refer to Willenborg et al. (2021).

Keywords: Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Metabolism; Molecular Biology; RNAseq; Sequencing; Single Cell.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Flow Cytometry
Leukocyte Count
Macrophages*
Mice
Sequence Analysis, RNA
Wound Healing*