Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT).
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.
Division of Immunology and Rheumatology, Stanford University School of Medicine, CCSR Building, Room 2215A, Mail Code 5166, 269 Campus Drive, Stanford, CA, 94305, USA.
D James
DJ
Haddon
Justin A
JA
Jarrell
Michael R
MR
Hughes
Kimberly
K
Snyder
Kelly M
KM
McNagny
Michael G
MG
Kattah
Paul J
PJ
Utz
D000954:Antigens, Surface / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008407:Mast Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000096:biosynthesis / Q000235:genetics; D013194:Staining and Labeling; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D000954:Antigens, Surface; D016207:Cytokines; D009841:Oligonucleotides; D014157:Transcription Factors