Murine Dermal Fibroblast Isolation by FACS

Journal of Visualized Experiments : JoVE
Graham G WalmsleyMichael T Longaker

Abstract

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expres...Continue Reading

Citations

Aug 15, 2020·Nature Communications·Deshka S FosterMichael T Longaker
Sep 3, 2020·Micromachines·Michael JanuszykGeoffrey C Gurtner
Oct 9, 2021·Proceedings of the National Academy of Sciences of the United States of America·Deshka S FosterMichael T Longaker
Apr 29, 2020·Dermatology : International Journal for Clinical and Investigative Dermatology·Helen StrandtSandrine Henri

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