Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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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. One-color experiment; Organism: Mus musculus; Custom Whole Genome Mouse 8x60k designed by Genotypic Technology Pvt. Ltd. (Agilent-026986); Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)

ORGANISM(S): Mus musculus

SUBMITTER: Genotypic technology

PROVIDER: E-GEOD-28517 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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