T Cell Protocols: Second Edition by Angus Stock, Vincenzo Cerundolo (auth.), Gennaro De Libero PDF

Biology

By Angus Stock, Vincenzo Cerundolo (auth.), Gennaro De Libero (eds.)

ISBN-10: 1588295877

ISBN-13: 9781588295873

ISBN-10: 1603275274

ISBN-13: 9781603275279

ISBN-10: 2008941671

ISBN-13: 9782008941677

With a large choice of investigative techniques, T mobile immunology is a crucial and open box of analysis. In T mobilephone Protocols, moment Edition, a world panel of specialists give a contribution totally up-to-date vintage protocols in addition to newly confirmed novel options for the learn of T lymphocyte biology. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, the chapters during this quantity supply short introductions to the subjects, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and Notes sections which acquire specialist tips about troubleshooting and averting identified pitfalls.

Up-to-date and simple to exploit, T telephone Protocols, moment Edition is a perfect consultant for younger investigators new to the advanced box of immunology in addition to a priceless, concise source for knowledgeable scientists trying to find transparent, efficacious descriptions of novel methods.

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Extra info for T Cell Protocols: Second Edition

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Key words: Flow cytometry, PBMC, phosphoprotein, immune cells, signaling, phospho-antibodies, activation, multiparameter, phosphorylation. 1. Introduction Flow cytometry is developing into an exciting tool for the study of post-translational modifications at the single cell level. The advances in hardware and software, in parallel with commercially available reagents enable analysis of signaling networks in primary samples such as peripheral blood mononuclear cells (PBMC). Highly specific fluorophore-conjugated antibodies are combined with flow cytometry to perform quantitative multiparameter analysis of single cells within complex cell population (1).

4. iNKT cell clones are grown at 37°C and 6% CO2. Growing iNKT cell clones are phenotyped for the expression of V24, V11 and CD4 coreceptor by flow-cytometry. 2. Expansion of iNKT Cell Polyclonal Cell Lines with the Specific Antigen GalCer In Vitro 1. PBMC are plated at 106 cells/ml at 24-well plates in 2 ml of complete medium containing 10–100 ng/ml of GalCer. After 48 h, 20 U/ml of rhIL-2 (Novartis, Emeryville, CA, US) are added. IL-2 is then added again at 40 U/ml every 48 h (see Note 1).

Include appropriate controls (at least vector control, untreated control) 2. Intravenous injections (tail vein injection) of the treatment group with 106 cfu E. coli expressing shRNA against CTNNB1 re-diluted in 200 ml PBS 3. Intravenous injections of the vector control group with E. , GFP). 4. Treat both groups three times per week for 3 weeks. 5. Analysis 1. RTqPCR to quantify and determine changes in the levels of target gene RNA. 2. Western blot to determine target gene (and downstream) protein concentration.

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T Cell Protocols: Second Edition by Angus Stock, Vincenzo Cerundolo (auth.), Gennaro De Libero (eds.)


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