| RRC ID |
38208
|
| Author |
Arakaki A, Ooya K, Akiyama Y, Hosokawa M, Komiyama M, Iizuka A, Yamaguchi K, Matsunaga T.
|
| Title |
TCR-beta repertoire analysis of antigen-specific single T cells using a high-density microcavity array.
|
| Journal |
Biotechnol Bioeng
|
| Abstract |
The antigen specificity of cytotoxic T cells, provided by T-cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single-cell level is required. In this study, we applied a high-density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR-beta gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent-labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T-cell population. The isolated single cells were then directly utilized for RT-PCR. By sequencing the amplified PCR products, antigen-specific TCR-beta repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single-cell level. This simple, accurate, and cost-effective technique for single-cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells.
|
| Volume |
106(2)
|
| Pages |
311-8
|
| Published |
2010-6-1
|
| DOI |
10.1002/bit.22663
|
| PMID |
20073087
|
| MeSH |
Cell Separation / instrumentation*
Equipment Design
Equipment Failure Analysis
Oligonucleotide Array Sequence Analysis / instrumentation*
Receptors, Antigen, T-Cell / genetics*
Reverse Transcriptase Polymerase Chain Reaction / instrumentation*
|
| IF |
4.002
|
| Times Cited |
10
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| Resource |
| Human and Animal Cells |
|
