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HIT analysis of T cells activated in the presence of TGF-β

ABSTRACT: We have developed a multi-analyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare human naive CD4+ T cells activated in the presence or absence of TGF-β. Keywords: response to stimulus

ORGANISM(S): Homo sapiens

PROVIDER: GSE10762 | GEO | 2008/03/08

SECONDARY ACCESSION(S): PRJNA107547

REPOSITORIES: GEO

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HIT: a new versatile protein array platform for multi-analyte phenotyping of surface molecules

2008-03-03 | GSE10665 | GEO

HIT analysis of T cells activated in the presence of TGF-Î²

Project description:We have developed a multi-analyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare human naive CD4+ T cells activated in the presence or absence of TGF-Î². Keywords: response to stimulus Two-condition experiment, with or without TGF-Î². Biological replicates: 3 normal human donors, each donor served as an internal control. One replicate per array. One of the biological replicates was performed as a dye-swap to monitor dye-bias.

2010-05-26 | E-GEOD-10762 | biostudies-arrayexpress

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Project description:We have developed a multi-analyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we screened 90 antibodies directed against a panel of cell surface markers as well as 4 isotype controls to compare resting human naive CD4+ T cells versus CD4+ T cells activated for 48 h with anti-CD3/anti-CD28 coated beads. Keywords: protein profiling, response to stimulus Two-condition experiment, activated versus resting cells. Biological replicates: 3 normal human donors, each donor served as an internal control. One replicate per array. One of the biological replicates was performed as a dye-swap to monitor dye-bias. We also performed a self-self comparison between activated cells from two different donors.

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Project description:We have developed a multi-analyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we coupled 44 of the tags to aliquots of an IgG1 isotype negative control antibody and the 4 remaining tags we coupled to anti-CD3, anti-CD4, anti-CD19, and anti-CD20 to create a 48-plex HIT cocktail. We then used this cocktail to stain 1 x 10^6 T or B cells and during amplification incorporated either cyanine 3-UTP (Cy3-UTP) or Cy5-UTP. Additionally, we snap froze and thawed an aliquot of the cocktail (FT). Keywords: protein profiling, cell type comparison

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