Cy3 is recognized as the dimmest fluorophore in the Cy dye family. To overcome this limitation, Cy3B was developed as an enhanced derivative, exhibiting a substantial increase in fluorescence quantum yield and photostability. Despite these improvements, Cy3B's limited aqueous solubility presents significant challenges for its efficient conjugation to biomolecules. In response, iFluor® 560 has been introduced, offering enhanced water solubility while retaining spectral properties closely aligned with both Cy3B and Cy3, positioning it as a robust alternative for the development of bioconjugates with bright orange fluorescence. It is optimally excited by 532 nm and 561 nm laser lines and is fully compatible with TRITC filter sets, facilitating its integration into existing experimental workflows. iFluor® 560 is suitable for labeling a wide range of antigens, including cell surface, intracellular, and intranuclear targets. It performs exceptionally well in complex multicolor panels, bridging the gap between fluorophores like FITC and PE. In simpler panels, iFluor® 560 can effectively replace PE to minimize emission spreading into PE tandem dyes. This dye allows for high molar ratio conjugation to proteins with minimal self-quenching, resulting in brighter conjugates. Its robust fluorescence quantum yield and photostability make iFluor® 560 ideal for detecting low-abundance biological targets, delivering greater precision and sensitivity in quantitative fluorescence assays.

The succinimidyl ester of iFluor® 560 is a widely used reagent for the conjugation of this dye to proteins or antibodies. Succinimidyl esters react selectively and efficiently with primary amines (such as the side chains of lysine residues or aminosilane-coated surfaces) at pH 7–9, forming stable covalent amide bonds. This property makes iFluor® 560 succinimidyl ester an excellent choice for labeling proteins, amine-modified oligonucleotides, and other amine-containing molecules.