tissue: primary endothelial cells from umbilical cord
Treatment protocol
Endothelial cells (ECs) were prepared from human umbilical cord veins by digestion with 0.5 mg ml-1 collagenase type I (Sigma, St. Louis, MO) for 15 min at 37 C as previously described, cells were used within 3 passages. S. aureus were harvested after overnight incubation at 37 ºC on blood agar plates and suspended to 0.5 McFarland in PBS (pH 7.4), corresponding to approximately 1 x 108 cfu ml-1. The infection of HUVEC with S. aureus was performed as previously described. After 4 h of incubation, supernatants were collected and 350 ul RLT-buffer (Qiagen GmbH, Hilden, Germany) was added to each well.
Growth protocol
37 degree, 5% CO2
Extracted molecule
total RNA
Extraction protocol
Rneasy
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 microg of cRNA were hybridized for 16 hr at conditions recommended by the manufacturer. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the Affymetrix GCS 3000.
Description
LJ0030
Data processing
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 150.
