What is intracellular cytokine staining?
Intracellular cytokine staining (ICS) is a method of cytokine analysis that provides information on the type of cytokines that are produced upon (antigen-specific) stimulation of T cells and B cells. Cytokine production can then be assessed via flow cytometry.
What is ELISpot assay used for?
Enzyme-linked immunosorbent spot (ELISpot) is a highly flexible assay and can be adapted to multiple readout formats. ELISpot assays are quantitative and measure key cellular functions of immune system cells. ELISpot has been used to assess both adaptive and innate immune responses.
How does an ELISpot work?
ELISPOT is performed using a PVDF or nitrocellulose membrane 96-well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody.
What types of cells can be used in ELISpot?
With the ELISpot, you can study antigen-specific cytokine responses, antibody specific secreting cells, tumor antigens, granzyme B and Perforin release by T cells, vaccine efficacy, epitope mapping, cytotoxic T-cell activity, detection of IL-4, IL-5, and IL-13, vaccine-induced antibody responses, antigen-specific …
How does PMA ionomycin work?
PMA activates protein kinase C, while ionomycin is a calcium ionophore, and stimulation with these compounds bypasses the T cell membrane receptor complex and will lead to activation of several intracellular signaling pathways, resulting in T cell activation and production of a variety of cytokines.
Can you stain for cytokines?
Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. The recipient cells are analyzed ex vivo after isolation from the peripheral blood following either nonspecific stimulation or donor-specific stimulation.
Which kind of antibodies are used in ELISpot assay?
ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate.
Are ELISpot substrates soluble?
ELISA substrates generate soluble products, while ELISpot substrates generate precipitating products. Several different soluble and precipitating substrates are available for both ALP and HRP enzymes.
How is the intracellular cytokine staining assay used?
Intracellular cytokine staining is a flow cytometry -based method for enumeration of antigen-specific, cytokine-secreting T cells.
How are cytokines detected in permeabilized T cells?
Because of the greater level of nonspecific binding inherent in fixed, permeabilized cells, greater care must be taken in designing specificity controls. A protocol in which brefeldin A is used to identify cytokine-producing T cells activated in response to in vivo contact with microbial antigens is also described.
How is flow cytometry used to detect cytokines?
Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. Multiparameter flow cytometry permits simultaneous detection of two or more cytokines within a single cell, allowing direct T (H)1 versus T (H)2 determination.
How is Brefeldin A used to detect cytokines?
A protocol in which brefeldin A is used to identify cytokine-producing T cells activated in response to in vivo contact with microbial antigens is also described. Animals