Description: The 8B.2C12 monoclonal antibody reacts with mouse CD366 (TIM3), a Th1-specific cell surface protein. CD366 is a type I transmembrane protein and contains an immunoglobulin and a mucin-like domain in its extracellular portion and a tyrosine phosphorylation motif in its cytoplasmic portion. CD366 is expressed selectively by differentiated CD4+ Th1 and CD8+ Tc1, but is absent on Th2 and Tc2. Other hematopoietic cell types, including naive T cells, B cells, macrophages and dendritic cells, do not express CD366, at least at the protein level. Expression of CD366 is upregulated at a late stage of T cell differentiation on Th1 cells after 3 rounds of in vitro polarization suggesting a role for this molecule in the transport or effector function of Th1 cells rather than a contribution to T cell differentiation. In an experimental autoimmune encephalomyelitis (EAE) model, CD366 was shown to be expressed on most CD4+ and CD8+ T cells in the central nervous system at the onset of clinical signs of disease, while less than 2% of CD4+ cells in the periphery expressed CD366 after immunization. In this model, in vivo administration of 8B.2C12 resulted in a hyperacute and atypical disease phenotype. It is postulated that the engagement of CD366 during T cell activation results in the expansion and activation of macrophages and increased severity of autoimmune disease. The Tim gene family may have an important role in the regulation of autoimmunity and allergies.

The 8B.2C12 antibody binds to the BALB/c allele of CD366 while reactivity to the C57Bl/6 allele is significantly weaker.

Applications Reported: This 8B.2C12 antibody has been reported for use in flow cytometric analysis.

Applications Tested: This 8B.2C12 antibody has been tested by flow cytometric analysis . This may be used at less than or equal to 0.125 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.

Super Bright 600 is a tandem dye that can be excited with the violet laser line (405 nm) and emits at 600 nm. We recommend using a 610/20 bandpass filter. Please make sure that your instrument is capable of detecting this fluorochrome.

When using two or more Super Bright dye-conjugated antibodies in a staining panel, it is recommended to use Super Bright Complete Staining Buffer (Product # SB-4401) to minimize any non-specific polymer interactions. Please refer to the datasheet for Super Bright Staining Buffer for more information.

Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 405 nm; Emission: 600 nm; Laser: Violet Laser

Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.