Expression and modulation of adhesion molecules on human bronchial epithelial cells

PGM Bloemen, MC Van den Tweel… - American journal of …, 1993 - atsjournals.org
PGM Bloemen, MC Van den Tweel, PAJ Henricks, F Engels, SS Wagenaar, AA Rutten…
American journal of respiratory cell and molecular biology, 1993atsjournals.org
Materials and Methods Cell Cultures The bronchial epithelial cell line BEAS-2B (16) was
obtained from Dr. 1. F. Lechner (National Institutes of Health, Bethesda, MD). The BEAS-2B
cell line is derived from human bronchial respiratory epithelium after transformation by
adenovirus 12-SV40 hybrid virus (16). BEAS-2B cells were maintained on plastic culture
flasks (Costar, Cambridge, MA) and coated for 2 to 6 h with a solution of 30 J. tg/rnl vitrogen
(Celtrix Laboratories, Palo Alto, CA), 10 J. tg/rnl fibronectin (Centraal Laboratorium …
Materials and Methods
Cell Cultures The bronchial epithelial cell line BEAS-2B (16) was obtained from Dr. 1. F. Lechner (National Institutes of Health, Bethesda, MD). The BEAS-2B cell line is derived from human bronchial respiratory epithelium after transformation by adenovirus 12-SV40 hybrid virus (16). BEAS-2B cells were maintained on plastic culture flasks (Costar, Cambridge, MA) and coated for 2 to 6 h with a solution of 30 J. tg/rnl vitrogen (Celtrix Laboratories, Palo Alto, CA), 10 J. tg/rnl fibronectin (Centraal Laboratorium Bloedtransfusiedienst, Amsterdam, The Netherlands), and 10 J. tg/ml bovine serum albumin (BSA)(fraction V; Boehringer Mannheim GmbH, Mannheim, Germany) in a serum-free keratinocyte medium (Keratinocyte-SFM; GIBCO, Grand Island, NY) with 50 J. tg/rnl gentamycin (GIBCO). The bronchial epithelial cell line NCI-H292 (American Type Culture Collection CRL 1848)(17, 18) was cultured in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum (FCS)(GIBCO) on uncoated culture flasks. Microscopically normal looking human bronchial tissue (third order or more), obtained from patients undergoing a thoracotomy, was used for culture according to the explant method of Lechner and LaVeck (19). However, keratinocyte medium was used instead of LHC-9 medium for cell culturing. In short, human bronchial tissue (from 12 donors) was cut into small fragments and placed in fibronectin/vitrogen/BSA-coated 6-well dishes (Costar). When the epithelial cell outgrowths reached confluence (after 2 to 3 wk), the bronchial tissue pieces were removed and placed in another culture dish. The tissue segments were transplanted to another dish no more than 2 times. The epithelial monolayer was used immediately for fluorescence experiments.
Endothelial cells (kind gift of Dr. R. 1. Benschop, Department of Immunology, Academic Hospital Utrecht, The Netherlands) were isolated from human umbilical cord vein and cultured in fibronectin-coated plastic culture flasks (Costar) using RPMI 1640 supplemented with 20% heatinactivated pooled human serum, penicillin (100 U/rnl), and streptomycin (100 J. tg/rnl). Cells were used at the fourth or fifth passage.
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