其他专刊

Protocols for Human Immune Monitoring

Flow cytometry and the more recently developed mass cytometry—or cytometry by time-of-flight (CyTOF)— are key technologies broadly used by researchers to study the immune system. Since its invention in the late 1960s, fluorescence-based flow cytometry has advanced considerably and now allows the simultaneous detection of 18-20 proteins expressed by individual cells. Fluorescence-based flow cytometry has enable researchers to better understand the complexity of the immune system. Yet, the phenotypic and functional heterogeneity of immune cells has trigger the need for even more proteins to be detected at once. To overcome the limitation imposed by the spectral overlap of fluorochromes, CyTOF technology uses elemental isotopes instead of fluorochromes to label antibodies. CyTOF enables simultaneous detection of 30-50 proteins and could, in theory, detect up to 100 markers. Using these technologies, immunologists have developed a broad range of staining procedures, gating strategies, and functional assays to better understand the immune system.

Our special issue on human immune monitoring presents a comprehensive collection of detailed and peer reviewed protocols focused on human sample processing for flow cytometry analysis (section 3), immune assays to assess the composition, phenotype and functionality of human immune cells by fluorescence-based flow cytometry (section 2) and by CyTOF technology (section 1). Bio-protocols’ uniquely interactive platform supports communication between scientists – through feedback, Q&A, and protocol updates sections – and will allow you to set up cytometry based technologies for your research. Bio-protocol is a living platform and our Protocols for Human Immune Monitoring special issue will grow with the cytometry field, giving you access to the latest developments.

专辑委员会 实验方案目录

In vitro Culture of Human PBMCs
人外周血单核细胞(PBMC)的体外培养
作者:Santosh K Panda and Balachandran Ravindran日期:02/05/2013,浏览量:101364,Q&A: 11

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) ...

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Isolation of Human PBMCs
人外周血单核细胞(PBMC)的分离
作者:Santosh K Panda and Balachandran Ravindran日期:02/05/2013,浏览量:85940,Q&A: 9

Peripheral blood mononuclear cells (PBMCs) are chiefly lymphocytes and monocytes. PBMCs are separated from the whole blood by a density gradient centrifugation method using Ficoll Histopaque.

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Whole Blood Staining of Human Monocyte Subsets for Flow Cytometry
应用于流式细胞术的针对单核细胞亚群的人全血染色
作者:Zheng Liu日期:05/20/2011,浏览量:40005,Q&A: 11

This is a general protocol to stain whole human blood for flow analysis with minimal spontaneous activation of monocytes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.

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Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
使用Vacutainer®细胞制备管(CPTTM)分离外周血单核细胞
作者:Alaina Puleo, Chantia Carroll, Holden Maecker and Rohit Gupta日期:01/20/2017,浏览量:31659,Q&A: 0

Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that ...

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Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
人淋巴细胞或外周血单核细胞(PBMC)的细胞因子胞内染色(ICS)
作者:Sheena Gupta and Holden Maecker日期:04/05/2015,浏览量:22812,Q&A: 1

Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but ...

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Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
适用于CyTOF质谱流式细胞仪的外周血单核细胞(PBMC)细胞因子胞内染色方法
作者:Dongxia Lin, Sheena Gupta and Holden Maecker日期:01/05/2015,浏览量:19392,Q&A: 3

In this protocol, we use a CyTOFTM mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium ...

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Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
采用CyTOF质谱流式细胞仪研究人外周血单核细胞表型
作者:Michael D. Leipold and Holden Maecker日期:01/20/2015,浏览量:15849,Q&A: 3

Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with ...

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Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
CyTOF质谱流式细胞仪测定外周血单核细胞中细胞因子刺激后的磷酸化变化
作者:Rosemary Fernandez and Holden Maecker日期:06/05/2015,浏览量:15063,Q&A: 1

Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and ...

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Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
采用流式细胞术评估人树突细胞的抗原摄取
作者:Ana Luque, Sonia Cárdenas-Brito, Rut Olivar and Josep M. Aran日期:11/20/2013,浏览量:15008,Q&A: 0

Antigen uptake by dendritic cells is the first key step towards induction of antigen-specific T-cell responses. This flow cytometry-based protocol describes the analysis of dendritic cell uptake of soluble antigens through two different mechanisms: non-specific macropinocytosis (using Lucifer Yelloy CH), and receptor-mediated endocytosis (using DQ ...

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Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation
人抗原特异性T淋巴细胞的体外纯化和脱粒
作者:Willemijn Hobo, Wieger Norde and Harry Dolstra日期:12/05/2012,浏览量:13038,Q&A: 0

Proliferative capacity and degranulation are important features of antigen-specific CD8+ T cells. By combining tetramer staining with a CFSE staining, we were able to enumerate the total number of antigen-specific T cells, as well as their number of divisions upon antigen-specific stimulation during a week. In addition, we performed ...

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Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry
CyTOF质谱流式细胞仪测定全血中细胞因子刺激形成的磷酸化变化
作者:Rosemary Fernandez and Holden Maecker日期:06/05/2015,浏览量:12088,Q&A: 1

The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single ...

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Cryopreservation of Human Serum
人血清的低温贮藏
作者:Yael Rosenberg-Hasson and Holden Maecker日期:11/05/2015,浏览量:7678,Q&A: 0

This protocol describes how to collect and aliquot human serum and store at -80 °C for future cytokine/chemokine/protein profile analysis.
Please use personal protective equipment (PPE) as required when handling samples with potential Bloodborne Pathogens.

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Determination of Cellular Uptake and Endocytic Pathways
细胞摄取和内吞途径的检测
作者:Jing Gong, Hong-Xia Wang and Kam W. Leong日期:02/20/2019,浏览量:6512,Q&A: 0

Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is ...

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Cell Barcoding in CyTOF (Inviting)
日期:12/30/2014,浏览量:145,Q&A: 0

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