Isolation and Phenotypic Characterization of Lung Fibroblasts
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Primary fibroblasts represent a heterogeneous population of cells that can be separated into subsets on the basis of cell
surface markers such as Thy-1. Deriving fibroblasts initially involves obtaining tissue explants from tissues such as the
lung, heart, cornea, skin, and orbit. The tissue is mechanically dissociated and cells are allowed to proliferate from the
fragments. Following establishment of a primary culture of fibroblasts, it is necessary to characterize the new strain of
cells to ensure their purity and fibroblastic phenotype using immunofluorescence and immunohistochemistry to detect the presence
or absence of cell-specific surface markers. Characterizing the cells as expressing or lacking Thy-1 can also be performed
by immunofluorescence in concert with microscopy or by flow cytometry using an anti-human Thy-1 antibody. In addition, fibroblasts
may be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting and/or magnetic beading; use of
these techniques can yield greater than 99% purity. Once separated, the pure Thy-1 expressing or lacking fibroblast subsets
can be propagated. These subsets can then be used for experimentation to determine functional differences between fibroblasts
derived from normal and pathological tissue such as scarred lung.
Affiliation(s): (4) Department of Environmental Medicine, University of Rochester, Rochester, NY
(5) Department of Obsterics and Gynecology, University of Rochester, Rochester, NY
(6) Department of Ophthalmology, University of Rochester, Rochester, NY
(7) Division of Pulmonary and Critical Care Medicine, University of Rochester, Rochester, NY
(8) Division of Molecular Medicine, Harbor-UCLA Medical Center, Torrence, CA
(5) Department of Obsterics and Gynecology, University of Rochester, Rochester, NY
(6) Department of Ophthalmology, University of Rochester, Rochester, NY
(7) Division of Pulmonary and Critical Care Medicine, University of Rochester, Rochester, NY
(8) Division of Molecular Medicine, Harbor-UCLA Medical Center, Torrence, CA
Book Title: Fibrosis Research: Methods and Protocols
Series: Methods in Molecular Medicine | Volume: 117 | Pub. Date: Aug-08-2005 | Page Range: 115-127 | DOI: 10.1385/1-59259-940-0:115
Subject: Molecular Medicine
Key Words: Fibroblasts - flow cytometry - fibrosis - lung - Thy-1 - magnetic beading - heterogeneity - fluorescence-activated cell sorting
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