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Costimulation through costimulatory molecules (CD40 or CD86) Synergizes with TLR-2 in the Activation of Resting B Cells: A Novel BCR Independent Approach of B Cell Stimulation

ABSTRACT: Resting B cells were isolated from mice splenocytes. Briefly, single cell suspension was prepared and treated with B cell enrichment cocktail supplemented with biotinylated anti-CD43 Abs. The cells were then treated with same volume of streptavidin-magnetic beads and negatively selected on BD IMagnet. The purity of RB cells obtained was >98%, as established by flow cytometry (CD45R^+ CD19^+ CD43^- CD4^- CD8^- ). Purified cells were cross linked with anti-CD86 Abs or anti-CD40 Abs, washed and cultured for 4 hours in the presence or absence of Pam2CSK4. Cells were harvested and washed and treated with RNAlater and subjected to microarray analysis. Microarray was performed by Genotypic Technology Pvt. Ltd. (Bangalore, India; www.genotypic.co.in ). The samples for gene expression were labeled using Agilent Quick-Amp labelling kit (p/n5190-0442). The labeled cRNA samples were hybridized on to a Genotypic designed Custom Whole Genome Mouse 8x60k (AMADID No: 26986). Cy3-labeled samples (800 ng) were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the gene expression hybridization kit of Agilent. Hybridization was carried out in Agilent's Surehyb Chambers at 65 C for 16h. The hybridized slides were washed using Agilent gene expression wash buffers and scanned using the Agilent microarray scanner, G2505C at 3 resolution. Data extraction from images was done using Feature extraction software v 10.5.1.1 of Agilent. Feature extracted data were analyzed using GeneSpring GX v 11 software from Agilent. Genes were classified based on the functional category and pathways using GeneSpringGX Software and Genotypic Biointerpreter-Biological Analysis Software.

ORGANISM(S): Mus musculus

PROVIDER: GSE28517 | GEO | 2013/12/31

SECONDARY ACCESSION(S): PRJNA138963

REPOSITORIES: GEO

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Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65°C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturer’s instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed student’s t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%.

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Costimulation through costimulatory molecules (CD40 or CD86) Synergizes with TLR-2 in the Activation of Resting B Cells: A Novel BCR Independent Approach of B Cell Stimulation

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