ELISA: The purified MP2-8F8 antibody (Cat. No. 554381) is useful for measuring mouse IL-3 protein levels when utilized as a capture antibody in sandwich ELISA. Purified MP2-8F8 antibody can be paired with the biotinylated MP2-43D11 antibody (Cat. No. 554384) as the detecting antibody, with recombinant mouse IL-3 (Cat. No. 554579) as the standard. Purified MP2-8F8 antibody should be titrated 2-6 µg/mL to determine the optimal concentration for ELISA capture. To obtain linear standard curves, doubling dilutions of mouse IL-3 (Cat. No. 554579), ranging from ~2000 to 15 pg/mL, are recommended for inclusion in each ELISA plate. When measuring mouse IL-3 in serum or plasma, investigators are encouraged to utilize BD OptEIA™ Mouse IL-3 Set (Cat. No. 555228).
Note: This ELISA pair has been reported to show no cross-reactivity with the following the cytokines:
Mouse IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 p70, IL-15, GM-CSF, IFN-γ, MCP-1, TCA-3, TNF
Human IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 p70, IL-12 p40, IL-13, IL-15, G-CSF, GM-CSF, IFN-γ, lymphotactin, MCP-1, MCP-2, MIP-1α, MIP-1β, NT-3, PDGF-AA, sCD23, SCF, TNF, LT-α, VEGF; rat IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF).
Neutralization: The NA/LE™ MP2-8F8 antibody (Cat. No. 554379) has been reported to be useful for neutralization of mouse IL-3 bioactivity.
Flow cytometric analysis and blocking control for intracellular staining: The MP2-8F8 antibody is useful for flow cytometric analysis in identifying and enumerating IL-3 producing cells within mixed cell populations. PE-conjugated MP2-8F8 antibody (Cat. No. 554383) is especially suitable for these studies. In addition, purified MP2-8F8 antibody can be used as a blocking control to demonstrate specificity of IL-3 staining by PE-conjugated MP2-8F8 antibody. To perform this control, the fixed/permeabilized cells (~1 million) can be incubated with 1 - 10 µg of unlabeled MP2-8F8 antibody (Cat. No. 554381) for 20 minutes at 4°C, prior to staining with PE-MP2-8F8. The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.