normalization data transformation protocol
Intensity data were extracted using Agilent's Feature Extraction (FE) software (version 220.127.116.11) including a quality control based on internal controls using Agilent's protocol GE1_107_Sep09. All chips passed the quality control and were analyzed using the Limma package 43 of Bioconductor 44. ID_REF = VALUE = processed Cy3 signal intensity (Agilent gProcessedSignal)
array scanning protocol
Cy3 intensities were detected by one-color scanning using an Agilent DNA microarray scanner (G2505B) at 5 micron resolution. Scanned image files were visually inspected for artifacts and then analyzed.
The hybridizations were performed for 17 hours at 10 rpm and 65°C in the hybridization oven (Agilent). Washing and staining of the arrays were done according to the manufacturer's recommendation.
Microarrays were done using the "Low RNA Input linear Amplification Kit Plus, One Color" protocol (Cat. N°: 5188-5339, 2007; Agilent Technologies, Inc., Santa Clara, CA) and the Agilent RNA Spike-In Kit for One color (Agilent Technologies, Inc. 2007; Cat. N°: 5188-5282) following the manufacturer's standard protocol. Global gene expression analysis was applied using the Agilent SurePrint G3 Human GE v2 8x60K Kit, P/N G4851B, (Agilent Microarray Design ID 039494. 200 ng of total RNA were used as a starting material to prepare cDNA. cDNA synthesis and in vitro transcription (IVT) were performed according to the manufacturer's recommendation. Quantity and efficiency of the labeled amplified cRNA were determined using the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
nucleic acid extraction protocol
RNA extraction, including a DNAse treatment step, was performed with RNeasy Micro (QIAGEN, Hilden, Germany) following manufacturer’s instructions.
TCCs from CSF-infiltrating T cells sorted after staining with specific TCR Vβ-familes, were established by seeding in 96-well U-botton microtiter plates 0.3 T cells cells/well with 2 x 105 non-autologous, irradiated PBMC, 2.5 µg/ml of PHA-L (Sigma) and IL-2 at the same medium used to expand CSF cells. Additional IL-2 was added every 3-4 days and after two weeks cells were pooled and analyzed, cryopreserved or restimulated.
sample treatment protocol
1 x 106 T cells were stimulated with 1 µg/ml of surface-coated anti-CD3 (OKT3, Ortho Biotech Products, Raritan, NJ) and 10-7 M PMA (SIGMA) for 24h.