1μg/ml LPS (Escherichia coli type 055:B6; Sigma) was added to the medium at day 6.
Growth protocol
Peripheral blood mononuclear cells (PBMCs) were isolated and CD14+ monocytes were sorted by positive selection using magnetic beads (Miltenyi Biotec, Germany). The cells were then cultured for 5-7 days in RPMI 1640 supplemented with 1000 U/ml GM-CSF and 1000U/ml IL-4 (PeproTech, Rocky Hill).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from moDCs using the miRNeasy mini kit (Qiagen, Germany).
Label
Cy3
Label protocol
Labeled cRNA were purified by RNeasy mini kit.
Hybridization protocol
Each slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent Technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent Technologies, Santa Clara, CA, US), according to the manufacturer's instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent Technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, PMT 100%, 20bit. Data were extracted with Feature Extraction software.
