The biological activities of IL-12 are mediated through a high-affinity receptor complex composed of the IL-12 receptor beta 1 (IL-12 Rbeta1) and IL-12 receptor beta2 (IL-12 Rbeta2) subunits. IL-12 Rbeta2 is a 130kD protein that interacts with p35 and is associated with Jak2. The expression of IL-12 Rbeta2 is absent on nai?ve T cells. Activation of T cells via TCR upregulates IL-12 Rbeta2 expression on human Th1, but not Th2 cells.
Applications:
Suitable for use in Flow Cytometry. Other applications not tested.
Recommended Dilution:
Flow Cytometry: 10ul labels up to 1x10e6 cells. Tested using human Th1-differentiated PBMCs. Briefly, PBMCs were treated with recombinant human IL-12 (5ng/ml) and anti-human IL-4 (10ug/ml) for 2 days, then activated with PMA (50ng/ml) plus ionomycin (200ng/ml) for 3 hours.
Optimal dilutions to be determined by the researcher.
Flow Cytometry Protocol:
1. Cells may be Fc-blocked with 1ug of human IgG/10e5 cells for 15 minutes at RT. Do not wash excess blocking IgG from this reaction.
2. After blocking, add 10ul of I8434-50C to up to 1x10e6 cells and incubate for 30 minutes at RT.
3. Remove unbound antibody by washing the cells twice in Flow Cytometry Staining Buffer (Catalog # F4568-10). Note that whole blood requires a RBC lysis step at this point using Flow Cytometry Human Lyse Buffer.
4. Resuspend the cells in Flow Cytometry Staining Buffer for analysis by flow cytometry. As a control for this analysis, cells in a separate tube should be treated with APC-labeled mouse IgG1 antibody.
Storage and Stability:
May be stored at 4 degrees C before opening. DO NOT FREEZE! Stable at 4 degrees C as an undiluted liquid. Stable for at 12 months at 4 degrees C. Freezing allophycocyanin conjugates will result in a substantial loss of activity. Allophycocyanin conjugates are sensitive to light.