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For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes (including BD OptiBuild Brilliant reagents) are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794).
The 500A2 monoclonal antibody specifically binds to the 25-kDa ε chain of the T-cell receptor-associated CD3 complex expressed on mouse thymocytes, mature T lymphocytes, and NKT cells. Plate-bound and soluble forms of the 500A2 antibody can activate T cells in vitro. Activation of a mouse T-cell clone by the 500A2 antibody can be blocked by Fab fragments of the GK1.5 anti-CD4 antibody. This suggests that the 500A2 antibody may bind an epitope on CD3e close to a site at which CD4 associates with the T-cell receptor. The 500A2 antibody reportedly does not to crossreact with rat leukocytes.
The antibody was conjugated to BD Horizon™ BUV737 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is a tandem fluorochrome of BD Horizon BUV395 with an Ex Max of 348-nm and an acceptor dye with an Em Max at 737-nm. BD Horizon Brilliant BUV737 can be excited by the ultraviolet laser (355 nm) and detected with a 740/35 filter. Due to the excitation of the acceptor dye by other laser lines, there may be significant spillover into channels detecting Alexa Fluor® 700-like dyes (eg, 712/20-nm filter).
Due to spectral differences between labeled cells and beads, using BD™ CompBeads can result in incorrect spillover values when used with BD Horizon BUV737 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BUV737 reagents (eg, CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
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