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[Methods in Molecular Biology] Immunological Tolerance Volume 1899 (Methods and Protocols) || Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting
摘要： Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4+CD25+CD127lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25+ PBMC, which reduces the duration of the subsequent FACS isolation.
关键词： FACS,Fluorescence-activated cell sorting,Regulatory T cells,Cell isolation,Treg
更新于2025-09-23 15:22:29
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An Optically Induced Dielectrophoresis (ODEP)-Based Microfluidic System for the Isolation of High-Purity CD45neg/EpCAMneg Cells from the Blood Samples of Cancer Patients—Demonstration and Initial Exploration of the Clinical Significance of These Cells
摘要： Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally de?ned as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical signi?cance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of ?uorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a micro?uidic system was proposed. In this study, an ODEP micro?uidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP micro?uidic system capable of simply and effectively isolating a speci?c, rare cell species from a cell mixture.
关键词： optically induced dielectrophoresis (ODEP),cancer metastasis,cell isolation,micro?uidic systems,circulating tumour cells (CTCs)
更新于2025-09-10 09:29:36