PMNs and PBMCs were isolated from venous blood of each participant in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases as described in Methods. PMNs or PBMCs (107) were combined on ice with or without IgG- and C3bi-coated latex beads (IgG-C3bi LBs) (8 x 107) in tissue culture plates pre-coated with 20% normal human serum (12-well plates) and centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis. Following centrifugation, samples were either processed immediately (0 h) or plates were incubated at 37oC in a CO2 incubator for 3 h or 6 h.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the Qiagen RNeasy kit according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2004, Affymetrix).
Hybridization protocol
cRNA were hybridized on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array according to the standard Affymetrix protocol (Expression Analysis Technical Manual, 2004, Affymetrix). GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using an Affymetrix scanner.
Description
healthy subject
Data processing
The data were analyzed with Microarray Suite version 5.0 and GeneSpring 5.0. Normalization protocol as follows. 1. Data transformation: set measurements less than 0.0 to 0.0. 2. Per Chip: Normalize to a constant value of 150.0 (MAS scaling factor). 3. Per Gene: Normalize to median.
