Bone marrow was isolated from 6-8 week old C57BL/6J wild type and MyD88-deficient mice. Macrophages from these marrows were cultured in DMEM media supplemented with 10% FBS and 15% L929 conditioned media (a source for CSF-1) for 8 days (plated at 3 million cells per 10 cm cell culture dish). Cells were treated for 2 hours with either 10 ng/mL re-purified LPS (from E. coli O111), live E. coli bacteria (log phase; 1 bacteria per macrophage) or media only as a reference (Mock). High quality total RNA was purified using a combination of the TRIzol protocol (Invitrogen) and RNeasy columns (Qiagen) according to the manufacturer's instructions. RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies). Total RNA samples were reverse transcribed and differentially labeled with Cy3 and Cy5 dyes (Amersham) using the Atlas PowerScript Fluorescent Labeling Kit (BD Biosciences). Labeled cDNA samples were hybridized over night in a Genomic Solutions hybridization station (Perkin-Elmer). The hybridization buffer was 3xSSC, 50 mM Hepes buffer, 0.1% SDS, pH 7.0, 30 ng/mL salmon sperm DNA, 30 ng/mL poly-A DNA, 30 ng/mL mouse Cot-1 DNA. The microarray slides were scanned with a GenePix 4000B microarray scanner and images quantified with the GenePix Pro 3.0 software (Axon Instruments). All data analysis and statistical analysis was performed with BioArray Software Environment (BASE; Lund University, Sweden) and GeneSpring software (Silicon Genetics). TIFF images can be downloaded from http://pga.mgh.harvard.edu Keywords = Mus musculus, LPS, endotoxin, macrophages, MyD88
