title: Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions.
abstract: The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca; Boston Children's Hospital, Division of Gastroenterology, Harvard Medical School.
authors.forename: Achille
authors.initials: A
authors.lastname: Broggi
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca.
authors.forename: Clara
authors.initials: C
authors.lastname: Cigni
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca; Boston Children's Hospital, Division of Gastroenterology, Harvard Medical School; Humanitas Clinical and Research Center; ivan.zanoni@unimib.it.
authors.forename: Ivan
authors.initials: I
authors.lastname: Zanoni
authors.affiliation: Department of Biotechnology and Biosciences, University of Milano-Bicocca; Humanitas Clinical and Research Center; francesca.granucci@unimib.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Granucci
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D002469:Cell Separation; D017364:Collagenases / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism; D000078404:Epidermal Cells; D005434:Flow Cytometry / Q000379:methods; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D020784:Matrix Metalloproteinase 8 / Q000494:pharmacology; D051379:Mice; D012867:Skin / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012879:Skin Physiological Phenomena; D013535:Suspensions; D013820:Thermolysin / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D013535:Suspensions; D017364:Collagenases; C417249:collagenase 1; D013820:Thermolysin; D020784:Matrix Metalloproteinase 8