Agilent Technologies fluorescent linear amplification reaction
Hybridization protocol
Agilent Technologies
Scan protocol
Agilent Technologies
Description
Antibodies Antibodies against murine CD4, FasL, IL-4, IFN, TNF, CXCR4, Mac-1, B220, CD11c and CD8 were isolated from hybridoma cell lines. The alpha-GalCer tetramers were produced as previously described. Antibodies against murine NK1.1, CD3, CD25, LFA-1 and Streptavidin-PE-Cy7 were obtained from BD Bioscience (Heidelberg, Germany) Isolation and purification of cells by MACS and FACS sorting Isolation of spleen and liver lymphocytes, red blood cell lysis and tetramer stainings were performed as described elsewhere [28]. Magnetic cell sorting (MACS) with MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed according to manufacturer’s protocol to enrich cell populations by positive selection. Subsequently, enriched cells were sorted using a FACS-Diva (BD Bioscience, Heidelberg, Germany). All manipulations were performed at 4°C. Cells were sorted into tubes containing cold PBS, 0.1% BSA to avoid activation of cells. Viable cells were detected by staining with PI (propidium iodid) or DAPI (Diamidino-phenylindol). The purity of sorted fractions was verified visually and by FACS reanalysis. At least 5 × 105 cells were isolated by FACS sorting and further used for microarray analysis. FACS sorts were repeated four times and duplicate samples of each sorted cell type were used for two independent microarray studies. Preparation of RNA from single cell suspension Total RNA was isolated by the TRIzol® Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany). Briefly, cells were resuspended immediately after FACS sorting in 500 µl TRIzol, shock frozen and stored at –80°C. Cells were thawed and further processed for total RNA isolation as described by the manufacturer. The amount of RNA was determined by OD260/280 nm measurement and total RNA was purified by RNeasy (Qiagen, Hilden, Germany). The RNA integrity and the amount of total RNA were measured with a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany).
Data processing
Features were extracted with an image analysis tool version A.6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmics platform Resolver Version 5.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A two fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.05), robust and reproducible.
