The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Video Article Published on JoVE

Rothschild DE, Srinivasan T, Aponte-Santiago LA, Shen X, Allen IC : JoVE 111, (2016) doi:10.3791/54033, Published 18 May 2016

This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques […]

By |January 11th, 2017|Categories: Ex Vivo, Laboratory Techniques, Protocols||0 Comments

Evaluation of T Cell Function in Allergic Disease

Barker, B. Methods Mol Biol. 2013;1032:31-44. doi: 10.1007/978-1-62703-496-8_3.

T lymphocytes play positive and negative roles in the pathogenesis of allergic disease. Isolation and functional characterization of T lymphocyte subpopulations is an important aspect of understanding allergy models and allergy therapies. Measurement of the T cell surface proteins and T cell proliferation can provide insight into T cell activation. […]

By |July 8th, 2014|Categories: Ex Vivo, Protocols||0 Comments

Assessment of Oxidative Metabolism

Imbeault E, Gris D. Methods Mol Biol. 2013;1031:51-7. doi: 10.1007/978-1-62703-481-4_6.

Oxidative metabolism is one of the central physiological processes that regulate multiple functions in a cell including cell death and survival, proliferation, gene transcription, and protein modification. There are multitudes of techniques that are used to evaluate oxidative activity. Here, we summarize how to measure oxidative activity by […]

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Quantification of Neturophil Extracellular Traps

Quantification and Visualization of Neutrophil Extracellular Traps (NETs) from Murine Bone Marrow-Derived Neutrophils. Vong L, Sherman PM, Glogauer M. Methods Mol Biol. 2013;1031:41-50. doi: 10.1007/978-1-62703-481-4_5.

Neutrophils are one of the first cells to respond to an inflammatory stimulus, and are equipped with an assortment of antimicrobial and proteolytic enzymes to disarm and degrade bacterial pathogens. A novel mechanism of […]

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Mast Cells in Mouse Models of Allergic Disease

Isolation and Characterization of Mast Cells in Mouse Models of Allergic Diseases. Kovarova, M. Methods Mol Biol. 2013;1032:109-19. doi: 10.1007/978-1-62703-496-8_8.

After their activation, mast cells release a variety of bioactive mediators that contribute to characteristic symptoms of allergic reactions. Ex vivo analysis of mast cells derived from their progenitors or isolated from mice is an indispensable tool for the […]

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Protocols for Enumerating Mouse Eosinophils

Dyer KD, Garcia-Crespo KE, Percopo CM, Sturm EM, Rosenberg HF. Methods Mol Biol. 2013;1032:59-77. doi: 10.1007/978-1-62703-496-8_5.

Eosinophils are prominent in allergic diseases, and their effector functions are studied in numerous gene-deleted and transgenic mouse models. However, mouse eosinophils and human eosinophils are not structurally or functionally equivalent, and assays designed to evaluate the properties of human eosinophils may […]

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DC Isolation from Lymph Node

Macpherson and McCoy Laboratories

Digestion medium: RPMI 2% FBS 1mg/ml collagenase et 0.1 mg/ml DNase

RPMI 1140 (GIMCO-ref 31870-025)
FBS-Foetal Bovine Serum (GIMO ref 10270-106)
Collagenase Type IA (Sigma C9891-1G)
DNase I (Roche 11 284 932 001)

Stop medium: RPMI 2% FBS 2mM EDTA

EDTA 0.5M ph 8.0 (GIMCO 15575-038)

Complete medium: RPMI 10% FBS 10mM HEPES 0.05 mM 2-mercaptoethanol 2mM Glutamine, […]

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Regulatory Functions of Bone Marrow-Derived Macrophages

Evaluation of Classical, Alternative, and Regulatory Functions of Bone Marrow-Derived Macrophages. Davis BK. Methods Mol Biol. 2013;1032:79-89. doi: 10.1007/978-1-62703-496-8_6.

The role of macrophage subsets in allergic diseases in vivo is under current investigation. These cells perform sentinel functions in the lung, the skin, and the gastrointestinal mucosa. Their interface with environmental cues influences the initiation, progression, development, and resolution […]

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Evaluating B Cells: Bone Marrow Precursors

Evaluating B-Cells: From Bone Marrow Precursors to Antibody-Producing Cells. Rosado MM, Scarsella M, Cascioli S, Giorda E, Carsetti R. Methods Mol Biol. 2013;1032:45-57. doi: 10.1007/978-1-62703-496-8_4.

Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context, flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identification of B-cell subsets, but can […]

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Pulmonary Antigen Presenting Cells: Isolation, Purification, and Culture

Nakano H, Cook DN. Methods Mol Biol. 2013;1032:19-29. doi: 10.1007/978-1-62703-496-8_2.

Antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages comprise a relatively small fraction of leukocytes residing in lymphoid and non-lymphoid tissues. Accordingly, functional analyses of these cells have been hampered by low cell yields. Also, alveolar macrophages share several physical properties with DCs, and this […]

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