Current Protocols in Immunology最新文献

This unit describes the monitoring and quantification of cellular cytotoxicity using a non-radioactive and real-time cytotoxic assay. The extent of target-cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell-imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO₂. The basic protocol describes natural killer cell-mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody-dependent cell-mediated cytotoxicity (ADCC). The assay is also suitable for antigen specific T-cell cytotoxic assays. Until now, the standard chromium 51 (⁵¹Cr)-release assay has remained the sole sensitive assay but its major drawbacks include cost and hazard of handling radioactivity. The real-time cytotoxic assay is therefore an effective alternative providing a robust and sensitive assay that accurately monitors lysis of target cells over time. © 2017 by John Wiley & Sons, Inc.

This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term ‘iNKT’ derives from the fact that a large fraction of murine and some human NK marker+ T cells (‘NKT’) recognize the MHC class I–like CD1d protein and use an identical ‘invariant’ TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N-region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, ‘iNKT’ cells). iNKT are mostly CD4+ or CD4–CD8– (‘double negative’), although a few CD8+ iNKT can be found in some humans. Basic Protocol 1 and Alternate Protocol 1 use multi-color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high-speed FACS sorting of iNKT cells. Basic Protocol 3 explains functional analysis of iNKT. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A support protocol for secondary stimulation and rapid expansion of iNKT cells is also included. © 2017 by John Wiley & Sons, Inc.

Semi-invariant natural killer T (iNKT) cells are CD1d-restricted innate-like lymphocytes that recognize lipid agonists. Activated iNKT cells have immunoregulatory properties. Human and mouse iNKT cell functions elicited by different glycolipid agonists are highly conserved, making the mouse an excellent animal model for understanding iNKT cell biology in vivo. This unit describes basic methods for the characterization and quantification (see Basic Protocol 1) and functional analysis of mouse iNKT cells in vivo or in vitro. This unit also contains protocols that describe enrichment and purification of iNKT cells, generation of CD1d tetramer, and lipid antigen loading onto cell-bound and soluble CD1d for activation of NKT cell hybridomas. © 2017 by John Wiley & Sons, Inc.

The tumor necrosis factor (TNF) superfamily of cytokines plays critical roles in all aspects of the immune response. TNF and the lymphotoxins (LT), LTα and LTβ, are particularly important as major effector cytokines and mediators or lymphoid organ development. One of the classical methods for the measurement of TNF and LTα activity is by demonstrating their ability to lyse certain target cells. A detailed protocol for the measurement of this activity using a highly sensitive indicator cell line is presented. More recently, ELISA assays have been developed to measure the protein concentration of these cytokines in any type of biologic fluid. © 2017 by John Wiley & Sons, Inc.

Electroporation—the use of high-voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternative techniques. This unit describes electroporation of mammalian cells, including ES cells, for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer, as well as for DNA vaccination. © 2017 by John Wiley & Sons, Inc.

Mouse models are used extensively to study human health and to investigate the mechanisms underlying human disease. In the past, most animal studies were performed without taking into consideration the impact of the microbiota. However, the microbiota that colonizes all body surfaces, including the gastrointestinal tract, respiratory tract, genitourinary tract, and skin, heavily impacts nearly every aspect of host physiology. When performing studies utilizing mouse models it is critical to understand that the microbiome is heavily impacted by environmental factors, including (but not limited to) food, bedding, caging, and temperature. In addition, stochastic changes in the microbiota can occur over time that also play a role in shaping microbial composition. These factors lead to massive variability in the composition of the microbiota between animal facilities and research institutions, and even within a single facility. Lack of experimental reproducibility between research groups has highlighted the necessity for rigorously controlled experimental designs in order to standardize the microbiota between control and experimental animals. Well controlled experiments are mandatory in order to reduce variability and allow correct interpretation of experimental results, not just of host-microbiome studies but of all mouse models of human disease. The protocols presented are aimed to design experiments that control the microbiota composition between different genetic strains of experimental mice within an animal unit. © 2017 by John Wiley & Sons, Inc.

Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design. Protocols to quantify and maximize sensitivity by titration of reagents and optimization of instrument settings, as well as basic surface and intracellular cell staining, are included. © 2017 by John Wiley & Sons, Inc.

The method first described by Ouchterlony in 1948 is a classic and simple technique that permits evaluation and comparison of antibodies in animal or human sera directed against protein or complex carbohydrate antigens. It is a low-tech procedure that may provide information on the relative quantity of antibody activity and the nature of the antigenic epitopes in different preparations. © 2017 by John Wiley & Sons, Inc.

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. © 2017 by John Wiley & Sons, Inc.

On identifying a new monoclonal antibody, or in characterizing antibodies in sera evoked by disease or immunization, it is particularly informative to determine the serological class of the antibodies. The serological class of the protein is determined by the structure of the antibody constant region. Several methods of class or isotype determination are outlined in this unit: sandwich ELISA, electrophoresis, and immunofixation, or use of a variety of commercially available kits. Different purification schemes and approaches for enzymatic fragmentation of the antibodies depend on the class or isotype of the antibody, so this information streamlines these processes. © 2017 by John Wiley & Sons, Inc.