The ISA-3 monoclonal antibody reacts with human ICOS (Inducible COStimulatory molecule), also known as H4, CRP-1 and AILIM. ICOS is a T cell specific activation molecule and a third member of the CD28/CTLA-4 family. Human ICOS has a relative molecular mass of 55-60 kDa, composed of 27 kDa and 29 kDa chains. Human ICOS on activated T cells has potent costimulatory activity for T cell activation and is required for humoral immune responses, in particular for memory B cell and plasma cell generation. ICOS binds to its ligand, B7h/B7RP-1 expressed on activated APCs (antigen presenting cells) and on a number of inflamed peripheral tissues. Plate-bound ISA-3 is costimulatory for T cells and induces production of IL-4, IL-5, IL-10 and other cytokines, but not IL-2. ISA-3 has the same reactivity pattern and characteristics as F44. ISA-3 was generated against the human ICOS antigen. C398.4A, anti-mouse ICOS/H4 (catalog # 14-9949-82), was shown to cross-react with human ICOS but binds to an epitope different from ISA-3. C398.4A stains activated cells brighter than ISA-3; however, it also exhibits higher staining of non-activated human peripheral blood or isolated PBMC. To achieve the brightest staining of ICOS on activated human T cells, please use 13-9948-82 or 12-9948-42 rather than 11-9948-42.

Applications Reported: The ISA-3 antibody has been reported for use in flow cytometric analysis.

Applications Tested: The ISA-3 antibody has been tested by flow cytometric analysis of unstimulated and CD3/CD28-stimulated (3 days) human blood cells. This can be used at less than or equal to 5 µL (0.6 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

Our internal testing shows that NovaFluor Yellow 730 non-specifically stains B cells in SJL mice. Non-specific staining has not been observed in BALB/c or C57BL/6 mice. Other strains have not been tested. See the Antibody Testing Data for an example of this strain-dependent difference.

NovaFluor dyes are not compatible with DNA intercalating viability dyes. Do not use viability dyes such as propidium iodide, 7-actinomycin D (7-AAD) and DAPI. Invitrogen LIVE/DEAD Fixable Dead Cell stains are recommended for use with NovaFluor dyes.

Each NovaFluor conjugate or kit is shipped with CellBlox Blocking Buffer. Use this buffer whenever staining with NovaFluor conjugates, including single-color compensation controls using cells. Whenever possible, we recommend adding CellBlox Blocking Buffer to antibody cocktails/master mixes prior to combining with cells. Add 5 µL per sample (regardless of the number of NovaFluors in your panel) to use the antibody cocktail as intended. For single-color controls, use 5 µL of CellBlox Blocking Buffer per 100µL of cell sample containing 10^3 to 10^8 cells.

NovaFluor conjugates are based on Phiton™ technology utilizing novel nucleic acid dye structures that allow for engineered fluorescent signatures with consideration for spillover and spread impacts. Learn more

Excitation: 552 nm; Emission: 730 nm; Laser: 561 nm (Yellow) Laser