THP-1 macrophages were then either left unstimulated (control) or stimulated with 1ug/ml LPS (LPS-stimulated) from Escherichia coli O55:B5 for 2 hours (Sigma Chemical Co., St. Louis, MO, USA) to induce an acute inflammatory response. To confirm an induction of the inflammatory response in the THP-1 macrophages, TNF mRNA levels were measured in control and LPS-stimulated samples
Growth protocol
THP-1 cells (monocyte cell line) (ATCC , Manassas, VA, USA) were maintained in culture in RPMI 1640 medium containing 10% fetal bovine serum, 1mM sodium pyruvate , 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2. For differentiation of THP-1 cells into macrophages a protocol using conditioned media as previously described by Whatling et al was used. To confirm differentiation of THP-1 monocytes into macrophages CD11b expression was measured using FACS and quantitative polymerase chain reaction (qPCR) (Text S2).
Extracted molecule
total RNA
Extraction protocol
total mRNA was extracted using TRIZOL® reagent (Invitrogen, Carlsbad, CA, USA) and purified using the RNeasy Mini Kit following the manufacturer’s instructions and included a DNase treatment (Qiagen, Valencia, CA, USA). RNA concentration and quality were analyzed using Nanodrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) respectively.
Label
biotin
Label protocol
The RNA was then hybridized and scanned on the Affymetrix® Genechip® Human Exon 1.0 ST array (Affymetrix, Santa Clara, CA, USA) following manufacturer’s protocols at the Karolinska Institute microarray core facility.
Hybridization protocol
Standard Affymetrix
Scan protocol
Standard Affymetrix
Data processing
CEL files were RMA normalized and log2 transformed using the apt-probeset-summarize algorithm as implemented in Affymetrix Power Tools 1.12.0. Meta probe sets were selected from the Affymetrix defined core set of well-established genes. probe group file: HuEx-1_0-st-v2.r2.pgf meta-probeset file: HuEx-1_0-st-v2.r2.dt1.hg18.core.mps
