cell type: Human myelogenous leukemia (macrophage-like) cells cell line: THP-1
Biomaterial provider
ATTC
Treatment protocol
Treated with LPS for 6h: Purified LPS derived from the enterohemorrhagic E. coli serotype O111 was purchased from Sigma Chemical Co (St. Louis, MO). Cells were washed with cold PBS before stimulation with Stx1 (400 ng/ml) in RPMI-1640 + 0.5% FBS for 6h
Growth protocol
The cells were maintained in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), penicillin (100 U/ml), and streptomycin (100 ug/ml) at 37°C in 5% CO2 in a humidified incubator.
Extracted molecule
total RNA
Extraction protocol
Total RNA from macrophage-like THP-1 cells was isolated using the RNAqueous kit (Ambion, Austin, TX), and 20 ug of total RNA was processed for microarray analysis. Briefly, cDNA synthesis, in vitro transcription, labeling and fragmentation to produce the oligonucleotide probes were performed following the GeneChip manufacturer’s instructions (Affymetrix, Santa Clara, CA). The probes were first hybridized to a test array (Affymetrix) and then to the GeneChip human genome HG-U133 Array (containing 54,675 probe sets representing more than 39,000 transcripts derived from ~33,000 well-substantiated human genes) using the GeneChip Hybridization Oven 640. The chips were washed in a GeneChip Fluidics Station 400 (Affymetrix), and the results visualized with a Gene Array scanner using Affymetrix software. The data were analyzed using the following software: GeneSifter (VizX Labs, Seattle, WA), Significance Analysis of Microarrays (SAM; Stanford University, Stanford, CA), and Spotfire DecisionSite 9.0 (Spotfire, Inc., Somerville, MA).
Label
biotin
Label protocol
Performed by UTMB microarray core per manufacturer's instructions
Hybridization protocol
Performed by UTMB microarray core per manufacturer's instructions
Scan protocol
Performed by UTMB microarray core per manufacturer's instructions. The results visualized with a Gene Array scanner using Affymetrix software.
Description
Performed by UTMB microarray core per manufacturer's instructions
Data processing
Data were RMA normalized using GeneSifter (VizX Labs, Seattle, WA).
