Fresh normal donors peripheral blood mononuclear cells isolated by Ficool-Hypaque (TBD sciences) centrifugation were enriched for CD4+ T cells (>97% purity by FACS) by magnetic negatively selection (CD4+T cell Isolation Kit II, Miltenyi Biotec). For PHA/IL-2 stimulation, cells were cultured at a density of 2×106/well with human recombinant IL-2 (100 units/ml, Pepro Tech) and PHA (5µg/ml, Sigma). For CD3/CD28 costimulation, cells were cultured at a density of 0.5×106/well with polystyrene beads coated of CD3 and CD28 antibodies (Dynabeads CD3/CD28 T Cell Expander, Dynal). The ratio of beads to cells was 3:1.
Growth protocol
Cells were cultured in 6-well plates at 37℃, 5% CO2 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 20mM HEPES
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US)following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany).
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
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. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), followed 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=5μm, PMT 100%，10% ，16bit.
Description
Gene expression of resting CD4+T cells
Data processing
The scanned images were analyzed with Feature Extraction software (Agilent technologies, Santa Clara, CA, US)with default settings.
