iFluor® 488 succinimidyl ester (NHS ester) is an amine-reactive fluorescent dye that can be used to label the primary amines on proteins and antibodies (such as lysine residues), as well as other amine-containing biomolecules. For instance, it can be conjugated to a secondary antibody as part of a signal amplification system (Figure 1), or it can be conjugated to an amine-modified oligonucleotide as part of a fluorescence in situ hybridization (FISH) experiment (Figure 2).

iFluor® 488 has excellent photostability and superior brightness when compared to classic green-fluorescent dyes, such as fluorescein (FITC). In a comparative experiment, iFluor® 488 and FITC were conjugated to goat anti-mouse IgG and used to bind mouse anti-tubulin. It was found that the fluorescence intensity loss of FITC was more than double that of iFluor® 488 (68% vs 27%) over the same period of time (Figure 3). Thus, iFluor® 488 presents significant advantages in experiments where photostability or long exposure times are of concern.

iFluor® 488 has been validated across multiple instrument platforms, including fluorescence microscopy, flow cytometry and spectral flow cytometry.

Spectral Flow Cytometry
iFluor® 488 is compatible with next generation spectral flow cytometers, such as the Cytek Aurora, allowing for expanded multiplexing capabilities. PBMCs stained with iFluor® 488 anti-human CD24 HI45 conjugate showed strong signal in the Aurora’s B2-A channel (Figure 6, Bottom). Spectral analysis of CD4-labeled human peripheral blood cells further confirmed the unique emission profile of iFluor® 488 on a 4-laser Aurora system (Figure 6, Top).

An example panel designed with iFluor® dye technology can be found here. To design a custom panel for your specific instrument, please see the Spectral Flow Cytometry Panel Builder Tool.

iFluor® 488 (ex / em = 491 / 516 nm) has spectral properties similar to Alexa Fluor® 488 (ex / em = 494 / 517 nm), allowing for straightforward implementation into existing workflows (Figure 7, Figure 8).