Primary monocytes isolated from normal PBMC (Peripheral Blood Mononuclear Cells), harvested at 1 hour without LTB4 stimulation.
Biomaterial provider
Human blood was provided by Normal Blood Donor Service of The Scripps Research Institute. PBMC isolation, monocytes isolation and treatment were carried out by Johnson & Johnson Pharmaceutical R&D (San Diego, CA).
Treatment protocol
For LTB4 treatment, cells were seeded in 12-well plates at 2 ´ 106 /well in 2 ml Hank’s buffered salt solution (HBSS) containing 1% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were incubated at 37°C for 1 hour before being stimulated with 60 nM LTB4 (final ethanol concentration 0.019%) or 0.019% ethanol as vehicle control.
Growth protocol
Human blood from healthy donors was obtained through Normal Blood Donor Service of The Scripps Research Institute. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Chandler, AZ). Briefly, peripheral human blood was diluted 1:1 with cold PBS containing 2 mM EDTA and 30 ml was then loaded carefully on top of 15 ml of Ficoll-Paque Plus in a 50-ml tube. After centrifugation at 2000 rpm for 30 min in a Sorvall RTH-750 tabletop centrifuge, the PBMC layer was carefully transferred and washed 1 ´ with cold PBS containing 2 mM EDTA. The cells were washed two additional times, and centrifuged at 1100 rpm to remove platelets in the supernatant. Primary monocytes were then isolated from PBMCs using the Monocyte Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s protocols.
Extracted molecule
total RNA
Extraction protocol
Cells were harvested at various time points and total RNA was isolated using the RNeasy Mini Plus Kit (Qiagen, Valencia, CA).
Label
biotin
Label protocol
One round of T7 polymerase-based linear RNA amplification was performed by reverse transcription of RNA with a T7 promoter oligo(dT) primer, followed by in-vitro transcription using the RiboBeast 1-Round Aminoallyl-aRNA Amplification Kit (Epicentre, Madison, WI) as specified in the manufacturer’s protocol with the following modifications: SuperscriptII (Invitrogen, Carlsbad, CA) was used as the reverse transcriptase, and the RNeasy 96 block was used for purification (Qiagen, Valencia, CA). Following purification, the cRNA target was conjugated with biotin-ester (Biotium, Hayward, CA), purified again with the Qiagen RNeasy 96 block, and concentrated by Speed-Vac (Thermosavant, Waltham, MA). Biotin-labeled cRNA was fragmented at 94°C for 20 minutes and added to Codelink hybridization buffer A and B (GE Healthcare).
Hybridization protocol
Each cRNA was applied to duplicate Codelink microarrays (GE Healthcare) according to the manufacturer’s protocol, and hybridized at 37°C overnight. Slides were washed and stained with an Alexa555-streptavidin conjugate (Invitrogen).
Scan protocol
The slides were scanned at 532 nm with an Agilent G2565BA Microarray Scanner (Agilent Technologies, Palo Alto, CA) according to manufacture instructions.
Description
human primary monocytes, no stimulation at 1 hour.
Data processing
Fluorescence intensity for each feature of the array was obtained by using Codelink EXP v4.1 software (GE Healthcare). For each sample, intensities from duplicate chips (when available) are averaged and quantile-quantile normalized cross all chips. The value field in result table holds the normalized intensity.
