6 protocols
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
nucleic acid extraction protocol
Qiagen RNeasy Mini Kit
normalization data transformation protocol
Standard Affymetrix protocol
growth protocol
Stromal cells were obtained from human tonsils collected from children undergoing routine tonsillectomy, after informed consent was obtained. Tonsils were cut into pieces and flushed using syringe and needle. Cell suspension was treated with DNAse I (Pulmozyme, Roche, Neuilly sur Seine, France) and collagenase IV (Worthington, Freehold, NJ) followed by centrifugation on a discontinuous Percoll gradient (Amersham, Piscataway, NJ). LN-derived stromal cells called ÒRestoÓ were established from the 15%/25% Percoll interface by long-term culturing. Cells were initially allowed to adhere for 48 hours followed by elimination of nonadherent cells and culture in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% selected fetal calf serum (FCS; HyClone, Logan, UT), and penicillin/streptomycin (PS). To study the gene expression profile, Resto-6 stromal cells were harvested at passage 5. Cell cultures were performed in RPMI1640 supplemented with 10% foetal calf serum.