9 protocols
AccessionType
normalization data transformation protocol
Expression values were determined using Affymetrix GeneChip Command Console Software (AGCC) and Console Software (Expression Console). ID_REF = VALUE = MAS5.0 signal intensity
array scanning protocol
GeneChips were scanned using the GeneChip Scanner 3000.
hybridization protocol
cRNA was hybridized to GeneChip Mouse Genome 430 2.0 Arrays from Affymetrix according the manufacturers protocol.
labelling protocol
Total RNA was labeled using the GeneChip 3' IVT Express Kit
nucleic acid extraction protocol
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
growth protocol
Splenic CD4 T cells were stimulated under Th2 culture conditions for 5 days in vitro. The Th2 cells were further cultured in vitro for another 2 days in the absence of any exogenous cytokines. The cultured CD4 T cells were then restimulated with immobilized anti-TCR mAbs with IL-2 and anti-IL-4 mAbs for 5 days. This cycle was repeated three times. (Th2-4th cells)
sample treatment protocol
Th2-4th cells were collected and treated with control siRNA (AM4635; Applied Biosystems) or Gata3 siRNA (s66482; Applied Biosystems) using the Mouse T cell Nucleofector Kit (Amaxa) according to the manufacturer's protocol. Mock- or Gata3 siRNA-transfected Th2-4th cells were harvested after 24hrs, and stimulated with or without immobilized anti-TCR mAb (H57-597; 3 mg/ml) for 4hrs. Then cells were collected and resuspendet in the Trizol solution (GibcoBRL).
growth protocol
Splenic CD4 T cells were prepared using a magnetic cell sorter (AutoMACS; Miltenyi Biotec) yielding a purity of >98%. Where indicated, cells from C57BL/6 mice were stimulated with immobilized anti-TCR mAb (H57−597; 3 mg/ml) and anti-CD28 mAb under Th2-culture conditions for 2days in vitro. Th2 conditions; 25 U/ml IL-2, 100 U/ml IL-4. After culturing for 3 more days, the cells were harvested.
sample treatment protocol
Th2 culture cells were collected and treated with control siRNA (AM4635; Applied Biosystems) or Gata3 siRNA (s66482; Applied Biosystems) using the Mouse T cell Nucleofector Kit (Amaxa) according to the manufacturer's protocol. Mock- or Gata3 siRNA-transfected Th2 cells were harvested after 24hrs, and stimulated with or without immobilized anti-TCR mAb (H57-597; 3 mg/ml) for 4hrs. Then cells were collected and resuspendet in the Trizol solution (GibcoBRL).