The 5D3/CD338 monoclonal antibody specifically binds to an epitope of ABCG2 (BCRP1), a multi-drug resistance protein that is a member of ATP binding cassette (ABC) transporters. It is highly expressed on primitive stem cells as identified by the "side-population" (SP) phenotype. This SP phenotype is based on the efflux of fluorescent dyes such as Rhodamine 123 and Hoechst 33342. The expression of ABCG2 appears to be highly conserved as it has been identified in various species. Studies show that highly purified murine stem cells express BCRP1 mRNA and this expression declines sharply as the stem cells express CD34. The highest levels of BCRP1 mRNA expression have been seen in KDR+ human stem cells. ABCG2/BCRP1 was clustered as CD338 in the HLDA8 workshop.
The antibody was conjugated to BD Horizon™ BUV661 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 661-nm. BD Horizon Brilliant BUV661 can be excited by the ultraviolet laser (355 nm) and detected with a 670/25 filter and a 630 nm LP. Due to cross laser excitation of this dye, there may be significant spillover into channels detecting APC-like emissions (eg, 670/25-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 BUV661 reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different BUV661 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.