Immature DC (2 x 10^6 cells/ml) were resuspended in fresh medium supplemented with 10 ng/ml GM-CSF, and 500 µl/well were seeded in 48-well tissue culture plates (Nunc, Roskilde, Denmark). Lactobacillus acidophilus NCFM (final concentration 10 ng/ml) and Bifidobacterium bifidum Z9 (final concentration 40 ng/ml) were added both individually and in combination (100 µl/well). The cell cultures were incubated at 37 ºC in 5 % CO2. The cells were harvested after 10h of stimulation.
Growth protocol
Bone marrow from three male C57BL/6 mice (Charles River Breeding Laboratories, Portage, MI, USA) was flushed out from the femur and tibia and washed. 3 x 105/ml bone marrow cells were seeded into 10 cm Petri dishes in 10 ml RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% (v/v) heat-inactivated fetal calve serum supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), glutamine (4 mM), 50 µm 2-mercaptoethanol (all purchased from Cambrex Bio Whittaker) and 15 ng/ml murine GM-CSF (harvested from a GM-CSF transfected Ag8.653 myeloma cell line). The cells were incubated for 8 days at 37ºC in 5% CO2 humidified atmosphere. On day 3, 10ml of complete medium containing 15 ng/ml GM-CSF was added. On day 6, 10ml were removed and replaced by fresh medium. Nonadherent, immature DC were harvested on day 8.
Extracted molecule
total RNA
Extraction protocol
Murine DC were harvested after 10 h of stimulation, homogenised by QIAshredder (Qiagen, Ballerup, Denmark), and RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). RNA quality was verified by Bioanalyzer (Agilent, Santa Clara, USA), and the concentration was determined by Nanodrop (Thermo, Wilmington, USA).
Label
biotin
Label protocol
1 μg RNA per stimulation condition was converted into cDNA, and biotin-labeled aRNA was synthesized using the MessageAmpTM II-Biotin Enhanced Kit (Ambion, Austin, TX, USA) according to the manufacturers instructions.
Hybridization protocol
The aRNA was fragmented and hybridized to Affymetrix Gene Chip Mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, CA, USA) according to manufacturers instructions.
Scan protocol
The arrays were scanned according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA) using Affymetrix GeneChip Scanner 7G.
Description
Mouse_NCFM_Z9_2.CEL
Data processing
The microarray data was analyzed using R and Bioconductor (Gentleman et al. 2004). Raw probe intensities were normalized using qspline and expression index calculations were performed using rma (Irizarry et al. 2003;Workman et al. 2002). For statistical testing Two-Way ANOVA was performed and statistical significance was assessed by estimating False Discovery Rate (FDR) by a Monte Carlo approach.
