Sentrix beadchips array mouse wg 6 v2

To analyze the possible mechanisms involved in mouse CD4⁺CD25^high regulatory T-cells (Treg cells) expansion promoted by mobilized MPP (MPP), we evaluated the transcriptomes of activated Treg extracted from simple culture (Treg control group) or from co-culture with MPPs (Treg + MPP). Treg and MPP were sorted as previously described. Treg were cultivated for 3 days with anti-CD3 and anti-CD28 alone or together with freshly purified MPP. After 3 days, CD4⁺ cells (Treg) were sorted for extraction of RNA using RNeasy Micro kit (QIAGEN). Its quality was verified in an Agilent Bioanalyzer. Total RNA was amplified and converted to biotinylated cRNA according to the manufacturer’s protocol (Illumina TotalPrep RNA Amplification Kit; Ambion). Paired biological replicates (3 for each group) were hybridized to the Sentrix BeadChips Array mouse WG-6 v2 (Illumina) and gene expression analysis was performed using GeneChip Mouse Genome 430 2.0 arrays (Affymetrix), as recommended by the manufacturer.

RNA from 2x10⁶ DMSO- or ZM39923-treated expanded CD4⁺GFP⁺ cells of Foxp3-eGFP mice was generated using RNeasy Mini kit (QIAGEN) and its quality was verified in an Agilent Bioanalyzer. Total RNA was amplified and converted to biotinylated cRNA according to the manufacturer’s protocol (Illumina TotalPrep RNA Amplification Kit; Ambion). Two biological duplicates were hybridized to the Sentrix BeadChips Array mouse WG-6 v2 (Illumina). Heatmap representation of log2 expression data was generated using R and Treeview software using the Euclidean correlation similarity measure and complete linkage algorithm. Scatter plot was performed using a log2 representation.