Isolation and phenotypic characterization of lung fibroblasts.
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.
Baglole
Carolyn J
CJ
Department of Environmental Medicine, University of Rochester, NY, USA.
Reddy
Sireesha Y
SY
Pollock
Stephen J
SJ
Feldon
Steven E
SE
Sime
Patricia J
PJ
Smith
Terry J
TJ
Phipps
Richard P
RP
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology*; D005355:Fibrosis / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008168:Lung / Q000166:cytology* / Q000473:pathology; D008280:Magnetics; D008856:Microscopy, Fluorescence; D010641:Phenotype