Other special issues

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.

CyTOF

Determination of Cellular Uptake and Endocytic Pathways
Authors:  Jing Gong, Hong-Xia Wang and Kam W. Leong, date: 02/20/2019, view: 1286, 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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Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
Authors:  Rosemary Fernandez and Holden Maecker, date: 06/05/2015, view: 9955, 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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Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry
Authors:  Rosemary Fernandez and Holden Maecker, date: 06/05/2015, view: 7773, 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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Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
Authors:  Michael D. Leipold and Holden Maecker, date: 01/20/2015, view: 9990, 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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Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
Authors:  Dongxia Lin, Sheena Gupta and Holden Maecker, date: 01/05/2015, view: 13232, Q&A: 2

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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Flow Cytometry

Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
Authors:  Sheena Gupta and Holden Maecker, date: 04/05/2015, view: 13206, 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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Assessment of Human Dendritic Cell Antigen Uptake by Flow Cytometry
Authors:  Ana Luque, Sonia Cárdenas-Brito, Rut Olivar and Josep M. Aran, date: 11/20/2013, view: 10632, 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
Authors:  Willemijn Hobo, Wieger Norde and Harry Dolstra, date: 12/05/2012, view: 9260, 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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Whole Blood Staining of Human Monocyte Subsets for Flow Cytometry
Author:  Zheng Liu, date: 05/20/2011, view: 33180, Q&A: 10

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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Sample Processing

Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM)
Authors:  Alaina Puleo, Chantia Carroll, Holden Maecker and Rohit Gupta, date: 01/20/2017, view: 16880, 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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Cryopreservation of Human Serum
Authors:  Yael Rosenberg-Hasson and Holden Maecker, date: 11/05/2015, view: 4896, 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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Isolation of Human PBMCs
Authors:  Santosh K Panda and Balachandran Ravindran, date: 02/05/2013, view: 51844, Q&A: 4

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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In vitro Culture of Human PBMCs
Authors:  Santosh K Panda and Balachandran Ravindran, date: 02/05/2013, view: 76869, Q&A: 9

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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