cell type: peripheral CD19pos B cells activated by: by CpG2006, anti-CD40 and IL-4 for 2 days flow cytometric cell sorting: IL-10pos B cells
Biomaterial provider
Blood bank Charité Campus Mitte
Treatment protocol
B cells were purified magnetically from PBMCs of healthy donors using anti-CD19-coupled magnetic beads. Cells were cultured in RPMI 1640 culture medium supplemented with L-glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 µg/ml), 10 µmol/L mercaptoethanol and 10% heat inactivated fetal calf serum. All cell cultures were carried out at 37 °C in 5% CO2. After 48 h stimulation with 1 µg/ml anti-CD40, 10 ng IL-4 and 3 µg CpG2006, B cells were restimulated for 3 h with phorbol myristate acetate (PMA, 10 ng/ml) and ionomycin (1 µg/ml). Next, the cells were coated with a bivalent antibody matrix against CD45 and IL-10 and after 1 h, secreted IL-10 was detected with a 2nd, fluorescent IL-10 specific antibody and the cells were sorted by Aria FACSorter machine according to expression of IL-10 or not.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instruction, including the recommended DNase digest. The purity and integrity of RNA were assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and amount was checked with a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).
Label
Biotin
Label protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Expression Kit from Affymetrix according to the manufacturers instruction. Briefly, after total RNA extraction, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to each of the GeneChip arrays.
Hybridization protocol
The HG-U133A plus 2.0 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C rotating for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Scan protocol
Arrays were scanned with an Affymetrix GeneChip Scanner 3000, using the GCOS software version 1.4 from Affymetrix.
Description
Gene-expression profiling of primary human B cells secreting IL-10 following activation with anti-CD40, IL-4 and CpG for 2 days.
Data processing
Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PubMedID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All 2 chips of group 1 were compared to the 2 chips of group 2 and chips within one group were compared to each other in both directions. The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p-value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not calculated with GCOS – t-tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using strict Bonferroni corrected Welch t-tests between 4 SLR values of Experiment group vs. Baseline group and 4 SLR values within both groups (the latter 4 giving a mean SLR value of zero; p-value had to be <= 1.143E-07) or more than 50% of non-parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 3 or more of all 4 Change calls have to be in the same direction. Significantly differentially expressed genes, were filtered using both default parameter sets of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009. You can find these validation experiments in BioRetis without registering, using the public content and public login. Click on view single results, select any existing Analysis beginning with first 3 letters "SGU" and click on "Next". Click on "Chose an existing parameterset" and select "JRG_Increase" for increased or "JRG_Decrease" for decreased probesets and click on "Fill". At the bottom of the site check the box named "* use Bonferroni correction" and click on "Search only" to get the list of significantly changed increased or decreased probesets, respectively. The Affymetrix Latin Square dataset, consisting of 42 chips in 14 experiments with three replicates each were analyzed in BioRetis with all possible 3 vs. 3 chip comparisons (one direction).
