T细胞受体
单元格排序
T细胞
分子生物学
生物
链霉菌
抗原
细胞毒性T细胞
细胞生物学
作者
Simone Rhein,Neşe Çakmak-Görür
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 95-128
标识
DOI:10.1007/978-1-0716-2441-8_6
摘要
AbstractThe protocol describes the procedure of antigen-specific T cell generation and TCR identification for the use in adoptive T cell therapy. We describe two paths of generating antigen-specific T cells, first, T cell stimulation with autologous dendritic cells pulsed with antigen peptide, second, in vivo T cell stimulation with peptide or DNA by gene gun application in a suitable mouse model followed by in vitro enrichment of peptide-reactive T cells. Peptide-stimulated T cells are sorted by fluorescence-activated cell sorting for CD107α or IFNγ expression and subsequently isolated RNA is used in a 5′ rapid amplification of cDNA ends (RACE )-PCR specific for TCR for TCR chain identification. After retroviral cloning, it is re-expressed on human T cells to test its applicability in adoptive T cell therapy.Key wordsTCRT cell stimulationAntigen-specific T cellsT cell sortRACE PCRViral transduction
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