Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, 6001, Western Australia, Australia.
Treatment protocol
tissue: Epithelial cells were collected by bronchial brushing and cultured. media: For culture, cells were washed once in RPMI-1640 media and the cell pellet resuspended in bronchial epithelial basal media (BEBM, Clonetics, CA) supplemented with bovine pituitary extract (50 mM), insulin (5 mM), hydrocortisone (0.5 mM), gentamicin (0.001%, v/v), amphotericin B (0.0005%, v/v), retinoic acid (0.1 µM), transferrin (10 mM), triiodothyronine (6.5 µM), epinephrine (6.5 µM) and human recombinant epidermal growth factor (EGF: 0.5 µM). plating: The plate was incubated for 20 minutes (37C, 5% CO2) to allow the macrophages to adhere. The suspended epithelial cells were aspirated from the plate, and the macrophages removed using trypsin (0.25%) for subsequent analysis. passage: The cells were seeded into a culture vessel (25 cm2 growth surface area) pre-coated with a mixture of fibronectin, collagen and bovine serum albumin, and maintained at 37C in a humidified incubator. Twenty-four hours post-isolation, unattached cells were collected. These cells were reseeded into the same culture vessel, with fresh media containing Ultroser G (2% v/v; BioSepra, CA), a serum substitute. The collection and reseeding of viable unattached cells was repeated at both 48 and 72 hours post isolation. Subsequent cultures were fed every second day and were usually passaged every 13–16 days.
Extracted molecule
total RNA
Extraction protocol
RTotal RNA was extracted from epithelial cells using the QIAGEN RNeasy kit (Vic, Australia).
Label
biotin
Label protocol
For microarray hybridization, RNA was processed for hybridization to Affymetrix GeneChip® Human HG-U133A Arrays according to the manufacturer’s instructions.
Hybridization protocol
The standard hybridization protocol was used as recommended by Affymterix.
Scan protocol
GeneChips were scanned using the Affymetrix GeneArray Scanner 3000.
