Neutrophils were isolated from blood provided by unknown donors.
Extracted molecule
total RNA
Extraction protocol
neutrophil total RNA was isolated using Trizol (Invitrogen, Burlington, ON, Canada) according to the manufacturer's protocol
Label
biotin
Label protocol
cDNA was purified by phenol-chloroform extraction using phase lock gels (Brinkmann, Westbury, NY, USA), then transcribed in vitro for 16 h at 37°C by using the IVT Labelling Kit (Affymetrix, Santa Clara, CA, USA) to produce biotinylated cRNA. Labelled cRNA was isolated by using the RNeasy Mini Kit column (QIAGEN, Mississauga, ON, Canada).
Hybridization protocol
Fifteen µg of fragmented cRNA were hybridized for 16 h at 45°C with constant rotation, using a human oligonucleotide array U133 Plus 2.0 (Genechip, Affymetrix, Santa Clara, CA, USA). After hybridisation, chips were processed by using the Affymetrix GeneChip Fluidic Station 450 (protocol: EukGE-WS2v5_450). Staining was made with streptavidin-conjugated phycoerythrin (SAPE; Invitrogen, Burlington, ON, Canada), followed by amplification with a biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA, USA), and by a second round of SAPE
Scan protocol
Chips were scanned using a GeneChip Scanner 3000 G7 (Affymetrix, Santa Clara, CA, USA) enabled for High-Resolution Scanning. Images were extracted with the GeneChip Operating Software (Affymetrix GCOS v1.4). Quality control of microarray chips was performed using the AffyQCReport software
Description
Neutrophils were resuspended at a concentration of 25 x 106 cells/ml in Hank's balanced salt solution (HBSS; 37°C) containing 1% fetal bovine serum, 10 mM HEPES pH 7.4, 1,6 mM Ca2+ and no Mg2+. Adenosine deaminase (ADA; 0.1U/mL) was added to cell suspensions 20 min prior to stimulation. Where mentioned, PGE2, CGS 21680, RO 20-1724 and forskolin, dissolved in DMSO, were added to cell suspensions 10 min before stimulation. The final organic solvent concentration was identical in all samples and did not exceed 0.1% (v/v). Neutrophils were stimulated with a mixture of LPS (100 ng/mL), GM-CSF (1.4 nM), TNF-α (100 ng/mL), fMLP (100 nM) and IL-1β (30 nM) for 30 min at 37oC. These experiments were performed 5 times in identical conditions, each time with a different donor.
