title: Murine Dermal Fibroblast Isolation by FACS.
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 expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine.
authors.forename: Graham G
authors.initials: GG
authors.lastname: Walmsley
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Zeshaan N
authors.initials: ZN
authors.lastname: Maan
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine; Department of Surgery, John A. Burns School of Medicine, University of Hawai'i.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Hu
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: David A
authors.initials: DA
authors.lastname: Atashroo
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Whittam
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Dominik
authors.initials: D
authors.lastname: Duscher
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Ruth
authors.initials: R
authors.lastname: Tevlin
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Owen
authors.initials: O
authors.lastname: Marecic
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: H Peter
authors.initials: HP
authors.lastname: Lorenz
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine.
authors.forename: Geoffrey C
authors.initials: GC
authors.lastname: Gurtner
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine; longaker@stanford.edu.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Longaker
mesh_terms: D000818:Animals; D005109:Extracellular Matrix; D005347:Fibroblasts / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D051379:Mice; D012867:Skin / Q000166:cytology*