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Mast Cells pp 381-400 | Cite as

Measurement of Mast Cell Surface Molecules by High-Throughput Immunophenotyping Using Transcription (HIT)

  • D. James Haddon
  • Justin A. Jarrell
  • Michael R. Hughes
  • Kimberly Snyder
  • Kelly M. McNagny
  • Michael G. Kattah
  • Paul J. UtzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1220)

Abstract

Here we describe the application of a highly multiplexed proteomic assay, called HIT (high-throughput immunophenotyping using transcription), to analyze human mast cell surface antigens at rest and during stimulation. HIT allows analysis of up to 100 analytes, including surface antigens and intracellular phosphoproteins, transcription factors, and cytokines, in a single experiment. Briefly, anti-mouse monovalent Fab fragments are covalently conjugated with barcoded oligonucleotides to generate a panel of conjugates. The oligonucleotide-Fab fragment conjugates are bound to monoclonal primary antibodies, creating a cocktail of up to 48 unique barcoded primary antibodies. As few as 100,000 mast cells are stained with the cocktail and the barcodes of the bound primary antibodies are amplified by in vitro transcription with fluorescently labeled NTPs. The resulting barcoded transcripts are quantified using a microarray spotted with oligonucleotides that are complementary to the barcoded transcripts. Differences in levels of the barcoded transcripts correlate well with actual protein levels and are capable of detecting stimulation-dependent changes in protein levels. HIT is an invaluable, broad-spectrum approach for characterizing mast cell surface antigens, signaling molecules, transcription factors, and cytokines.

Key words

Mast cells HMC-1 Microarray Proteomics Immunoassay Multiplex Surface marker profiling Transcription Monoclonal antibody 

Notes

Acknowledgements

P.J.U. is supported by NHLBI Proteomics Contract 268201000034C, Proteomics of Inflammatory Immunity and Pulmonary Arterial Hypertension; 5 U19-AI082719, National Institutes of Health; 2 OR-92141, Canadian Institutes of Health Research (CIHR); a gift from the Floren Family Trust; and a gift from the Ben May Trust. D.J.H. is supported by a CIHR postdoctoral fellowship.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • D. James Haddon
    • 1
  • Justin A. Jarrell
    • 1
  • Michael R. Hughes
    • 2
  • Kimberly Snyder
    • 3
  • Kelly M. McNagny
    • 2
  • Michael G. Kattah
    • 4
  • Paul J. Utz
    • 1
    Email author
  1. 1.Division of Immunology and RheumatologyStanford University School of MedicineStanfordUSA
  2. 2.Department of Medical Genetics, The Biomedical Research CentreUniversity of British ColumbiaVancouverCanada
  3. 3.Department of Experimental Medicine, The Biomedical Research CentreUniversity of British ColumbiaVancouverCanada
  4. 4.Division of Gastroenterology, Department of MedicineUniversity of California San FranciscoSan FranciscoUSA

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