Microarray analysis of splenic CD11b+Gr-1+ cells

We extracted RNA of sorted splenic CD11b+Gr-1+ cells and performed microarray analyses. Total RNA was isolated using the manufacturer’s recommended protocol for RNeasy (Qiagen, Valencia, CA). Isolated RNA was quantified, and quality was assessed using an Agilent A 2100 BioAnalyzer with the RNA NanoChip (Agilent, Andover, MA). We used 2μg of total RNA to make single-stranded antisense cDNA with the NuGEN Technologies (San Carlo, CA) Ovation Biotin System in accordance with the manufacturer’s directions. Labeled targets were hybridized to Affymetrix (Santa Clara, CA) MOE430 2.0 GeneChip microarrays for 16h at 45°C. The arrays were washed and scanned according to Affymetrix standard protocols. We used microarrays to detail the global gene expression of CD11b+Gr-1+ cells from the control mice and the clarithromycin-treated mice, and identified distinct classes of up-regulated genes during this process. Splenic CD11b+Gr-1+ cells from the control mice and the clarithromycin-treated mice were sortd, RNA extraction from these cells was hybridized on Affymetrix microarrays.