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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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

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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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