Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 assay: treatment of test cells with [BF/S+L/Ep] at 0, 25, 50, 75, 100, 500 μg/ml resulted in a cell viability, on average, of 100%, 102%, 107%, 104%, 99% and 73%, respectively, as compared to the vehicle control (data not shown). Treatment at 100 μg/ml [BF/S+L/Ep] might cause some limited cytotoxicity, whereas the dosage of 75 μg/ml does not result in any cytotoxic effects. The final DMSO concentration reached 0.1% in each DC culture. An aliquot of a 0.1% DMSO solution in med- ium only was thus used as a vehicle (negative) control. LPS from Escherichia coli (serotype 055:B5) was pur- chased from Sigma (St. Louis, MO) and used for cell activation or quality control at 1 μg/mL. Flow cytometric analysis All cell samples were stained using a direct immuno- fluorescence staining method. For each step of the stain- ing, 2 × 105 cells were treated with specific antibodies for 30 min at 4°C in 45 μL of phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Test cells of each experimental group were first incubated with purified anti-CD16/CD32 antibody (mouse IgG2a, 93) for 10 minutes on ice. Fluorescein isothiocyanate (FITC)- or phycoerythin (PE)-labeled monoclonal anti- bodies were then used for staining of MHC class II (I-Ab, mouse IgG2a, 2G9), CCR4 (mouse IgG, 2G12), CCR5 (mouse IgG, HM-CCR5), CCR7 (mouse IgG2a, 4B12), CD40 (mouse IgG, HM40-3), CD80 (mouse IgM, 16-10A1), CD86 (mouse IgG, GL-1) and CD11c (mouse IgG, N418) on test cells. All antibodies were purchased from Biolegend (San Diego, CA). After cell samples were incubated with test antibody at 4°C for 40 min, cells were washed twice with PBS and fixed with 1% paraformaldehyde. Test cells were then analyzed in a Coulter EPICS XL flow cytometer (Beckman/Coulter). DNA microarray analysis for gene expression Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instruction and used to generate cRNA targets. A total of 7 μg of RNA from each sample was used to synthesize the first strand cDNA using T7-Oligo (dT) primer and T7 RNA polymerase by in vitro transcription (IVT) reaction. The biotinylated cRNA products were then cleaned up according to the Affymetrix protocol. An aliquot of 15 μg of RNA per sample was then hybridized to a Affymetrix gene chip, the mouse Genome 430 2.0 array, containing 45,101 pro- besets and variants from over 34,000 well-characterized mouse genes, using a standard protocol suggested by the Affymetrix menu. Images of the array signals were col- lected on Affymetrix scanners. A total of 8 hybridizations were performed for immature DCs, while each time point and treatment was analyzed in duplicates on sepa- rate chips. Standard Pearson correlation coefficients were Page 16 of 18 used to determine the consistency of gene expression in each replicate Affymetrix array. ∑(x − x)(y − y ) ∑(x − x)2∑(y − y )2 Pearson correlation value = Data was analyzed using Spotfire software, which includes algorithms that determine whether a gene is absent or present and whether the expression level of a gene in an experimental sample is significantly increased or decreased relative to a control sample. Changes in expression levels are presented as averages of log2 (BF/ S+L/Ep treatment)/(DMSO treatment). The microarray data have been deposited to the Gene Expression Omni- bus database at NCBI (GEO; http://www.ncbi.nlm.nih. gov/projects/geo/) under the accession number GSE19369. 2-D gel electrophoresis and image analysis Total cellular proteins of immature dendritic cells were prepared using 0.3 mL of sample buffer [7 M urea (Bio-Rad), 2 M thiourea, 4% CHAPS (Sigma), 10% 1,4- dithioerythitol (Merck, Frankfurt, Germany), 2% Phama- lyte 3-10] by vortexing for 1 h and then collecting the protein supernatants by 55,000 RPM and for 1 h. Protein concentration was determined by using a protein assay kit (Bio-Rad, Hercules, CA). 2-D gel electrophoresis was carried out using the Bio-Rad Protean IEF cell and Pro- tean electrophoresis cell system as described previously [10,45] with modifications. Digitalized gel images were analyzed with 2-D analysis software (PD Quest, Bio-Rad) as previously described [10]. The image with the highest number of spots was selected as the master gel. Automa- tically detected images of protein spots in test gels were then manually edited to include the low intensity spots and correct for spot artifacts. For the match set contain- ing images with pI 5-8, the spot volume (intensity inte- grated over the spot area) was normalized by the volume of total spots in the gel. The data was then exported to Microsoft Excel. Protein identification by MALDI-TOF-MS Protein identification was performed as described pre- viously [10]. Briefly, each gel slice was cut into small pieces with a scalpel. Reduction was achieved with 10 mM DTT at 57°C for 1 h. Alkylation reaction was per- formed with 55 mM iodoacetamide for 1 h at room temperature in the dark. Gel spots were then washed for 10 min alternately with 25 mM NH4HCO3 and acet- onitrile. Gel pieces were completely dried using a Speed Vac. The dried gel pieces were then immersed in three volumes of trypsin solution (V5111; Promega, Madison, WI), at 20 ng/mL in 25 mM NH4HCO3 (freshly diluted).