Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT).
Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT).
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通过转录高通量免疫表型 (HIT) 测量肥大细胞表面分子。
DOI：
10.1007/978-1-4939-1568-2_24
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Utz,PaulJ
中科院分区：
文献类型：
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作者：
Haddon,DJames;Jarrell,JustinA;Hughes,MichaelR;Snyder,Kimberly;McNagny,KellyM;Kattah,MichaelG;Utz,PaulJ
在这里，我们描述了一个高度多路复用的蛋白质组学检测，称为HIT（高通量免疫表型使用转录），分析人肥大细胞表面抗原在休息和刺激过程中的应用。HIT允许在单个实验中分析多达100种分析物，包括表面抗原和细胞内磷蛋白、转录因子和细胞因子。简言之，将抗小鼠单价Fab片段与条形码化寡核苷酸共价缀合以产生一组缀合物。所述多核苷酸-Fab片段缀合物与单克隆一抗结合，产生多达48种独特的条形码一抗的混合物。将少至100，000个肥大细胞用混合物染色，并通过用荧光标记的NTP体外转录来扩增结合的一抗的条形码。使用用与条形码化转录物互补的寡核苷酸点样的微阵列定量所得的条形码化转录物。条形码转录物水平的差异与实际蛋白质水平密切相关，并且能够检测蛋白质水平的刺激依赖性变化。HIT是表征肥大细胞表面抗原、信号分子、转录因子和细胞因子的非常有价值的广谱方法。
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.