E-MEXP-3955 - Transcription profiling by array of mouse bone-marrow-derived dendritic cells after treatment with human WNT1
Last updated on 16 July 2014, released on 15 August 2015
Bone marrow (BM) cells derived from femurs of C57BL/6 mice were used to generate BM-DCs. For this, BM cells (2x106 cells/10 ml) were seeded on 100 mm2 bacterial dishes (Greiner bio-one, Frickenhausen, Germany). DC culture media (IMDM, 5% [v/v] FCS [PAA, Coelbe, Germany], 100 U/ml penicillin, 100 ug/ml streptomycin [both Gibco, Paisly, UK], 50 uM beta-mercaptoethanol [Roth, Karlsruhe, Germany] 5% [v/v] of murine GM-CSF containing cell culture supernatant was replenished on days 3 and 6 of culture. Aliquots of DC cultures were treated with recombinant human 100 ng/ml WNT-1 (Biovision, Milpitas, CA) for 48h on days 7 or 8 of culture. Each 20 ug of total RNA derived from unstimulated BM-DCs treated for 48 h with WNT-1 (100 ng/ml) or left untreated was used for generation of fluorescecne-labeled cDNA probes (Alexa Fluor 555-aha-dUTP for untreated BM-DCs; Alexa Fluor 647-aha-dUTP for WNT-treated BM-DCs).
Prehybridized (5X SSPE,0.1% SDS, and 1% BSA) microarrays (Mouse Whole Genome OneArray Microarray v2; Phalanx Biotech, San Diego, U.S.A.) were hybridized with each 20 pmol of either fluorescence-labeled cDNA probe using OneArray Hybridization Buffer with 18% formamide for 16 h (Lucidea SlidePro Hybridizer; GE Healthcare, Munich, Germany) according to the OneArray hybridization protocol. Washed microarrays were analysed using a ScanArray 4000 system equipped with ScanArray 4.0 software (both Perkin-Elmer; Rodgau-Jugesheim, Germany) at 10 pixel size. Laser intensity (90-100%) and photomultiplier tube sensitivity (60-70%) were varied to optimize signal to background ratios. Derived images weere analyzed using the ScanArray 4.0 easyquant software module. Intensity data were normalized (loess normalization; MIDAS-2.19) and subjected to significance analysis (MEV 4.3) using appropriate modules of the TM4 microarray software suite. Changes in gene expression with p < 5% were used for subsequent analyses. Gene ontology annotations were performed with Panther software (Thomas et al., 2003).
transcription profiling by array, co-expression, ex vivo, stimulus or stress design