NovaFluor Dyes for Immunophenotyping

NovaFluor dyes are built using Phiton technology. This is a macrostructure labeled with small-molecule fluorophores. Unique fluorescent signatures are created to avoid cross-excitation between laser lines, a common problem with conventional labels, while the emission profiles are designed to avoid spectral spill over into other channels. These unique properties simplify high-dimensional panel design by unlocking previously unusable channels in current instrumentation and delivering higher resolution data for cleaner single-cell analysis and separation.

The Phiton structure lends stability, thus the NovaFluor dyes retain fluorescence intensity and spectral signature at long-term 4°C storage after staining and fixation.

NovaFluor dyes can be used in flow cytometry by themselves or with other dyes. NovaFluor dyes offer great utility in the replacement of dyes that are excited by multiple lasers and that also spillover to multiple detectors. For example, PE-eFluor 610 dye is excited by both the blue and yellow laser and therefore, when emitting signal, will be collected in associated detectors. Replacing PE-eFluor 610 dye with NovaFluor Blue 610 and NovaFluor Yellow 610 minimizes spillover into associated detectors. This allows for one additional marker. Detectors that may previously have gone unused can detect NovaFluor dyes because of the narrow excitation and tighter emission spectra.

NovaFluor dyes are incompatible with nucleic acid binding dyes, including PI, 7AAD, DAPI, and cell-permeant dyes, DyeCycle dyes, and SYTOX dead cell stains. LIVE/DEAD Fixable dead cell stains should be used for viability analysis.

Brightness is helpful when looking for dimly expressed antigens. Employing fluorophores that exhibit varying levels of brightness is advantageous for experiments involving multiple markers, as this allows for better signal resolution. Below is an example of swapping a bright, cross-excited dye for two spectrally cleaner dyes to allow accommodation of one more marker. 

Typical channel distribution of an 11-color panel involving traditional dyes compared to a 13-color panel achieved by incorporating NovaFluor dyes. Dyes were replaced or added based on Figure 3 and examining excitation/emission spectra. 

When designing a panel, it is essential to understand key fluorophore characteristics such as the relative brightness of fluorophores and the amount of fluorescence spread each fluorophore generates across non-primary detectors. This information is used when pairing antibodies with fluorophores (Table 1). Brightness of a fluorophore in its primary detector can be calculated using the stain index, where a higher stain index represents greater separation between positive and negative populations. The relative stain index for fluorophores is often represented visually, as seen in this Staining Index for Fluorophore Brightness, where the stain index was calculated and used to rank the relative indices of the fluorophores from dim to brightest.

The amount of fluorescence each fluorophore generates across non-primary detectors is referred to as spread of the dye. Because each fluorophore in a panel has the potential to emit fluorescence into non-primary detectors, it is important to minimize the impact of the spread of each fluorophore through careful selection of fluorophores. Fluorophore spread can be calculated by measuring the sum of the fluorescence produced in all non-primary detectors of the instrument. Lower spread results in greater resolution, which is also referred to as spectral cleanliness. To ensure optimal resolution, use fluorophores with narrow emissions and minimal cross-laser excitation.

Table 1. List of fluorophores properties.

Commonly used fluorophores were evaluated using the Cytek 5-laser Aurora spectral cytometer with 64 detectors. The list of properties for each fluorophore includes excitation and emission maximum wavelengths, the primary detector wavelength range, primary excitation laser, relative contribution to spread, and stain index. Human peripheral blood mononuclear cells (PBMCs) were labeled with an anti-human CD4 antibody conjugated to each fluorophore. The data was acquired with Cytek assay settings using a lymphocyte gate. The stain index of each fluorophore was calculated using data from its primary detector. The relative contribution to spread by each fluorophore is represented by the sum of fluorescence emission into the 64 non-primary detectors, and classified as low, medium, or high.