Cyanine3.5 NHS ester is a dye for labeling of oligonucleotides, peptides and proteins which contain amino-groups. For fluorescence imaging and other fluorescence-based biochemical analysis, it has been used to label biological molecules too. Cy3.5 dyes have enhanced fluorescence upon binding to proteins. The excitation peak and emission peak of Cy3.5 dye is around 550nm and 590nm respectively. Most derivatives of non-sulfonated cyanines have low aqueous solubility except for hydrochlorides of hydrazides and amines. For biomolecule labeling, the labeling reagent has low aqueous solubility, using of organic co-solvent to dissolve this molecular is necessary for efficient reaction. First, Cyanine dye should be dissolved in organic solvent and then added to a solution of biomolecule in appropriate aqueous buffer.
The ENU cells immersed in 0.3 mM Cy3.5 NHS ester with modified balanced salt solution (MBSS) showed that Cy3.5 NHS ester does not label the dead cells [1].
Reference:
1. XueYi Xie. Tumor angiogenesis, O2 saturation, glucose and amino acid metabolisms study using functional imaging. PhD thesis.