NovaFluor dyes are designed for more resolution with narrow emission spectra and minimal cross-laser excitation. Lower spectral spillover or overlap lessens the need for compensation, decreases spreading error, and increases opportunities to add new markers. This aids in construction of flow cytometry panels with increased resolution while expanding the overall size of panels.
Benefits to adding NovaFluor dyes include:
Flow cytometry is used in a broad range of applications including immunophenotyping, fluorescent protein detection, rare event analysis, cell health characterization, and more. In this presentation we will review the basics of fluorescence and how to use a fluorescence excitation/emission spectrum, compare conventional vs. full spectrum flow cytometry, and review numerous types of fluorophores listing advantages and challenges of each.
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.
Figure 2. Process for generating a Phiton-labeled antibody. Fluorophore brightness can be engineered for precise separation index values. Phiton conjugation to an antibody allows 1:1 labeling for quantitative measurement.
Spectral flow cytometry panel builder tutorial
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.
Figure 3. Two multicolor flow cytometry panels designed for the Attune NxT Flow Cytometer. By including NovaFluor dyes in the panel (lower section of the table above), researchers are able to collect data on an additional 2 markers. By replacing PE and its tandems with the specific NovaFluor dyes, cross-laser excitation is removed, allowing for full use for blue and yellow lasers.
Animation of how spectral flow cytometry enables researchers to get more information about their cells in a single flow cytometry experiment.
Invitrogen CellBlox Blocking Buffer is formulated to block nonspecific binding of Invitrogen NovaFluor labels with cells. These nonspecific interactions can result in higher background labeling. CellBlox Blocking Buffer is a non-antibody, non-protein–based blocking solution, and should be used every time a NovaFluor dye is used for labeling any cell type to minimize background labeling (Figure 4).
CellBlox Blocking Buffer is also recommended for use with cyanine-based dyes or cyanine-based tandem dyes to block non-specific interactions with monocytes and macrophages to minimize background labeling (Figure 5).
Use of CellBlox Blocking Buffer requires minimal change to most flow cytometry staining protocols. Add 5 µL CellBlox Blocking Buffer directly to a cell suspension containing 103-8 cells prior to the addition of an antibody, with 100 µL as a final staining volume. CellBlox Blocking Buffer may instead be added to an antibody cocktail mixture prior to labeling cells, by adding 5 µL CellBlox Blocking Buffer for every stained sample to be labeled with the antibody cocktail mixture, with 100 µL as a final staining volume.
See how to use CellBlox Blocking Buffer in the Cell Surface Staining Protocol.
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.
|Fluorescent label||Excitation and emission min/max (nm)||Primary detector* (nm)||Laser line (nm)||Spread**||Stain index*|
|NovaFluor Blue 510||496/511||B1 (498–518)||488||Low||25.5|
|Alexa Fluor 488||495/519||B2 (516–533)||488||Low||61.8|
|Kiravia Blue 520™||495/520||B2 (516–533)||488||Medium||113.1|
|NovaFluor Blue 530||509/530||B2 (516–533)||488||Low||11.1|
|Spark Blue 550™||516/550||B3 (533–550)||488||Low||30.1|
|Alexa Fluor 532||532/554||B3 (533–550)||488||Low||7.6|
|NovaFluor Blue 555||494/555||B3 (533–550)||488||Low||10.4|
|NovaFluor Blue 585||494/585||B4 (571–590)||488||Low||19.9|
|NovaFluor Blue 610 / 30S||509/614||B6 (605–625)||488||Low||29.6|
|NovaFluor Blue 610 / 70S||509/614||B6 (605–625)||488||Low||46.3|
|NovaFluor Blue 660 / 40S||509/665||B7 (652–669)||488||Low||36.2|
|NovaFluor Blue 660 / 120S||509/665||B7 (652–669)||488||Medium||54.5|
|PerCP Vio 700||482/704||B9 (688–707)||488||Medium||33.2|
|PerCP-eFluor 710||482/710||B10 (707–727)||488||High||144.5|
|NovaFluor Yellow 570||552/568||YG1 (567–587)||561||Low||51.6|
|PE||496/578||YG1 (567–587)||488; 561||High||420.7|
|CF® 568||562/583||YG1 (567–587)||561||Medium||180.1|
|PE-Dazzle 594||566/610||YG3 (605–625)||488; 561||High||279.1|
|NovaFluor Yellow 610||552/612||YG3 (605–625)||561||Medium||116.0|
|NovaFluor Yellow 660||552/663||YG4 (652–669)||561||Medium||95.8|
|PE-Cy5||496/667||YG5 (669–687)||488; 561||High||539.3|
|NovaFluor Yellow 690||552/690||YG6 (687–706)||561||Low||68.1|
|PE-Cy5.5||482/695||YG7 (706–735)||488; 561||High||339.6|
|NovaFluor Yellow 700||552/700||YG7 (706–735)||561||Low||93.0|
|NovaFluor Yellow 730||552/731||YG7 (706–735)||561||Medium||125.0|
|PE-AF700||566/719||YG7 (706–735)||488; 561||Medium||81.5|
|PE-Cy7||496/785||YG9 (765–795)||488; 561||High||352.0|
|Alexa Fluor 647||650/688||R2 (669–687)||640||High||337.3|
|NovaFluor Red 660||637/659||R2 (669–687)||640||Low||88.5|
|NovaFluor Red 685||637/685||R3 (688–707)||640||Low||113.2|
|Spark™ NIR 685||665/685||R3 (688–707)||640||Medium||175.9|
|NovaFluor Red 700||639/700||R3 (688–707)||640||Low||138.8|
|NovaFluor Red 710||639/710||R4 (707–727)||640||Medium||105.5|
|Alexa Fluor 700||702/723||R4 (707–727)||640||Low||77.9|
|APC-eFluor 780||633/780||R7 (772–795)||640||Low||138.5|
|APC/Fire™ 750||650/787||R7 (772–795)||640||Low||180.9|
* All measurements were taken on a 5-laser Cytek Aurora
** Spread numbers are classified as Low for values <4,000, Medium for values 4,000–8,000, and High for values >8,000.
APC/Fire, PE/Dazzle, Spark Blue, are trademarks trademarks of BioLegend, Inc. CF® is a registered trademark of Biotium. KIRAVIA Blue 520™ is a registered trademark of SONY Corporation. NovaBlue, NovaYellow, NovaRed, and NovaFluor are registered trademarks of Phitonex, Inc.
NovaFluor CD4 Label Characterization Kits are formulated to enable the testing of all available NovaFluors as direct conjugates of CD4. These kits can be used to evaluate performance of NovaFluors on flow cytometers.
For Research Use Only. Not for use in diagnostic procedures.