Cells were stimulated for 24 hours with interferon- (IFN; 100 units/ ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml)
Growth protocol
SPeripheral blood samples were diluted with phosphate buffered saline (PBS), then underlaid with Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ), and centrifuged under standard conditions to separate mononuclear cells. PBMCs were removed and plated in nonsupplemented Cellgro RPMI 1640 (Mediatech, Herndon, VA) at 37°C for 2 hours. Following removal of nonadherent cells and media, the attached cells were cultured in R-10 (day 0), which consisted of RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), HEPES (Sigma, St. Louis, MO), penicillin/ streptomycin (Gibco), and L-glutamine (Fisher Scientific, Pittsburgh, PA), and which was supplemented with recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF; 100 units/ml) (PeproTech, Rocky Hill, NJ). Every other day, 75% of the medium was removed and fresh R-10 supplemented with GM-CSF was added.
Extracted molecule
total RNA
Extraction protocol
Cells were suspended in TRIzol (Invitrogen, Carlsbad, CA) and processed according to the manufacturer's instructions
Label
biotin
Label protocol
Affymetrix standard labeling
Hybridization protocol
Affymetrix standard
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip® Scanner 3000 7G.
Description
Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1-43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte-macrophage colony-stimulating factor.
Data processing
Data were imported into GeneSpring 7.3 with RMA preprocessing and normalized to median of all samples. Normalized data were exported and subjected to Distance Weighted Discrimination (DWD) to account for any batch effects then reimported into Gene Spring and analyzed.
