A collection of biochemical, cellular, and molecular techniques for unraveling and quantifying the events occurring between the initial contact of a cytokine at the membrane receptor and the eventual activation of gene transcription. The techniques used include the generation of transfectants, the immunohistochemical detection of cytokines in tissue sections, and optimized staining for cytoplasmic detection. Highlights include RT-PCR of small amounts of mRNA, in situ hybridization, biosensor analysis, measurement of biological activities and standardization, immunohistochemical and single-cell detection, and receptor isolation, characterization, and crystallization. Enjoy a quick and smooth introduction to the key methods used in cytokine research Use readily reproducible techniques that ensure successful experimental results Employ antisense-RNA, RT-PCR of small amounts of mRNA, and in situ hybridization.
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acetate activity affinity amino acid analysis analyte antibody apoptosis approx BIAcore binding Biol biological biosensor biotinylated cDNA cell line cellular centrifuge chemokines chemotactic chemotaxis chromatography clone coli column competitor complex concentration containing crystals culture cytokine cytotoxicity detection diluted dose–response electrophoresis elution enzyme ethanol expression filter gene granulocytes human IgG1 immobilized Immunol Incubate injection interaction interferon interleukin kinetic ligand medium membrane Methods mg/mL HSA monocytes mRNA NaCl necrosis factor Note nuclear protein PBS-S pellet peptide PGE2 phosphopeptide pipet plasmid plasmid DNA plates potency preparation primer probe production protein purified Quantitative reaction reagent recombinant resin Resuspend Rinse room temperature RP-HPLC sensor chip sensorgram sequence serum SH2 domain Sigma signal slides sodium soluble receptors solution staining standard store at 4°C Subheading substrate supernatant test samples tion tissue TNFSF transfection Tris-HCl tube tumor necrosis tumour necrosis factor vector virus vitro wash buffer