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Cytokine Bioassays (eBioscience)
Biological activity of cytokines and their concentrations are commonly measured by cellular proliferation of primary cell cultures or isolated cell lines that are dependent and/or responsive to a specific growth factor. Other aspects of biological activity of cytokines include induction of further cytokine secretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxis, and promotion of colony formation. In vitro assays to measure all of these activities are available. Here are general protocols for setting up bioassays to measure.
Immunofluorescence Staining and Flow Cytometry of Intracellular Cytokines(eBioscience)
A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly.
Cytokine Expression Profiling (eBioscience)
ELISPOT (Enzyme-linked ImmunoSPOT) technique was originally used to enumerate antibody secreting B cells. In the current technique, cells are deposited onto a membrane coated with one antibody specific for a protein followed by an appropriate incubation period. Subsequently, the protein of interest is detected in the environment immediately surrounding the cell secreting it, with another antibody specific for a different epitope of the protein. The signal detected by the HRP enzyme/substrate results in a colorimetric footprint of the cells and can be quantitated by visual scoring or specialized plate-readers.
T-Cell Activation Using mAb to CD3 (eBioscience)
This protocol is written as a starting point for examining in vitro proliferation of splenic T-cells stimulated via CD3. All the steps where optimization by the user can be achieved for meaningful results have been highlighted. Briefly, these include titration of responding cells, titration of the activating reagents and the optimal culture period for the best activation response. MTT assay is used for the detection of cellular proliferation.
Cytokine Bioassays Using Neutralizing mAbs (eBioscience)
Antibodies that block binding of cytokines to their specific receptors and neutralize their effects are critical in studies of cytokine function. The following three neutralization protocols describe in vitro bioassays using anti-mouse and anti-human cytokine antibodies. In general, the cytokine bioassay protocols are modified to pre-incubate the cytokine of interest with the specific neutralizing antibody prior to addition to the responding cells.
FAQs for Signal Transduction (LTI)
Lots of useful information on signal transduction
