The M1/69 monoclonal antibody specifically binds to CD24 (Heat-Stable Antigen, HSA or HsAg), a variably glycosylated, glycosyl-phosphatidylinositol-anchored membrane protein expressed on erythrocytes, granulocytes, monocytes, lymphocytes, and neurons. Hematopoietic stem cells of the embryonic yolk sac and fetal liver express CD24. Levels of expression of CD24 vary during differentiation of the T and B cell lineages. In the bone marrow, hematopoietic progenitors acquire CD24 expression upon commitment to the B-lymphocyte lineage. Immature B cells in the bone marrow express low CD24 levels whereas peripheral B lymphocytes express intermediate to high levels of CD24. The level of CD24 expression has been reported to rise upon activation of splenic B cells with LPS, but not with CD154 (CD40 Ligand). The majority of thymocytes express high levels of CD24, while most mature thymic and peripheral T lymphocytes do not express CD24. In contrast, TCR-bearing thymocytes which emigrate to the spleen are CD24+. Dendritic cells of the thymus, spleen, liver, and epidermal Langerhans cells have also been reported to express CD24. CD24 is not expressed by NK cells, as determined by staining with J11d mAb (Cat. No. 553146). CD24 is involved in the costimulation of CD4+ T cells by B cells, it is a "co-inducer" of in vitro thymocyte maturation, and it is a ligand of CD62P (P-selectin). While the monoclonal antibodies 30-F1, M1/69, and J11d all react with CD24, they show subtle differences in the level of staining of different lymphocyte populations. When possible, investigators should continue to use the same monoclonal anti-CD24 antibody as used in previous studies.
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.