NovaFluor Dyes for Immunophenotyping

Fluorescent dyes engineered to have narrow excitation and emission profiles

NovaFluor Dyes for Immunophenotyping

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:

  • Unique spectral signatures
  • Designed with narrow emissions and minimal cross-laser excitation
  • Decreased spillover spread for higher resolution
  • Stable dyes that can be stored long-term without losing fluorescence as compared to tandem dyes
  • Easy to add to existing panels

On demand webinar:

Fluorophore Fundamentals for Flow Cytometry

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 dye technology

Figure 1. Excitation ranges of NovaFluor dyes compared to PE-Dazzle 594 dye.

Normalized absorbance spectra of PE/Dazzle 594, NovaFluor Blue 610, and NovaFluor Yellow 610 with blue (488 nm), green (532 nm), yellow (561 nm), and red (637 nm) laser lines overlaid. From the data we observe that PE-Dazzle 594 signal is collected in two detectors. NovaFluor dyes exhibit narrower excitation ranges, so swapping PE-Dazzle with NovaFluor Blue 610 and NovaFluor Yellow 610 frees an additional channel for adding another marker.

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.

Figure2-Phiton-tech

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.


Using NovaFluor dyes in flow cytometry

Spectral Flow Cytometry Panel Builder

Choosing the optimal combination of fluorochromes can be simplified with a guided method. The Invitrogen Flow Panel Builder offers a customizable panel building process to fit your flow cytometry experimental needs, whatever your experience level.

 

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

Two multicolor flow cytometry panels designed for the Attune NxT Flow Cytometer

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.

Why are researchers excited about spectral flow cytometry?

Animation of how spectral flow cytometry enables researchers to get more information about their cells in a single flow cytometry experiment.

"Going away from the tandem paradigm cleans up the annoyances with designing panels in terms of better resolution and brighter markers. This is a steppingstone in the progress of cytometry. Now that we have tools like NovaFluor dyes, we might be able to get away with not getting a UV line that’s super expensive and still get the same number of really good markers.”

David LeClerc

- Technical Director at Cytometry and Antibody Core (CAT) Facility, University of Chicago

“The NovaFluor dyes all show more restricted excitation/emission profiles than traditional fluorophores in the same range and make for good replacements in our panels. The NovaFluor Blue 610, NovaFluor Blue 660, and NovaFluor Yellow 570 are also much “cleaner” than the respective versions.”

Oliver Burton, Ph.D.

- Staff Scientist, Babraham Institute


Using CellBlox Blocking Buffer with NovaFluor dyes

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.

  • Always use CellBlox Blocking Buffer with NovaFluor dyes when labeling cells for best background reduction
  • CellBlox Blocking Buffer is compatible with all fluorophores and with Invitrogen LIVE/DEAD Fixable Dead Cell Stains
  • CellBlox Blocking Buffer can be used with any fluorophore-antibody conjugate as a high-performance monocyte and macrophage blocking solution
  • CellBlox Blocking Buffer is compatible with other blocking reagents, such as Fc Block, blocking proteins, Brilliant Stain Buffer, and Super Bright Complete Staining Buffer
  • CellBlox Blocking Buffer is not required when labeling antibody-capture beads

See how to use CellBlox Blocking Buffer in the Cell Surface Staining Protocol.

Figure 4. CellBlox Blocking Buffer mitigates nonspecific interactions of NovaFluor dyes when using cells.

Peripheral blood mononuclear cells (PBMCs) were either unlabeled (grey) or labeled with CD3 Monoclonal Antibody (clone UCHT1), NovaFluor Yellow 660 (Cat. No. H002T03Y04) with (red) and without (blue) the addition of CellBlox Blocking Buffer (Cat. No. B001T06F01). Forward Scatter vs. Side Scatter density plot shows lymphocyte and monocyte gating (A). Histogram overlay plots of CD3 expression are shown using a lymphocyte gate (B) and a monocyte gate (C). CD3 labeling combined with CellBlox Blocking Buffer is shown to reduce background in lymphocytes and reduce non-specific labeling of monocytes and macrophages, as compared with CD3 labeling without CellBlox Blocking Buffer, leading to an improvement in resolution. Data were acquired on a 4-laser Invitrogen Attune NxT Flow Cytometer using the 561 nm laser with a 695/40 nm bandpass filter.

Flow cytometry using CellBlox Blocking Buffer and NovaFluor Dyes

Figure 5. CellBlox Blocking Buffer mitigates nonspecific interactions of Cyanine tandem dyes when using cells.

Peripheral blood mononuclear cells (PBMCs) were labeled with a CD14 direct conjugate of APC and a CD3 direct conjugates of APC-Cyanine 7, PE-Cyanine 7, and PerCP-Cyanine 5.5, with and without the addition of CellBlox Blocking Buffer (Cat. No. B001T06F01). Forward Scatter vs. Side Scatter density plot shows a combined lymphocyte and monocyte gate (A). Dot plot overlay plots of CD3 and CD14 expression display cell labeling with (red) and without (blue) CellBlox Blocking Buffer. Use of CellBlox Blocking Buffer is shown to reduce nonspecific interactions with monocytes and minimize background labeling of cells with PE-Cyanine 7 (B), APC-Cyanine 7 (C), and PerCP-Cyanine 5.5 (D) tandem dyes. Data were acquired on a 4-laser Invitrogen Attune NxT Flow Cytometer using a 488 nm laser with a 695/40 nm bandpass for PerCP-Cyanine 5.5, a 561 nm laser with a 780/60 nm bandpass filter for PE-Cyanine 7, and a 638 nm laser with a 780/60 nm bandpass filter for APC-Cyanine 7.

Flow cytometry using CellBlox Buffer and Cyanine tandem dyes

Selecting the best fluorophore

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
BB515 490/515 B1 (498–518) 488 High 254.9
Alexa Fluor 488 495/519 B2 (516–533) 488 Low 61.8
FITC 494/520 B2 (516–533) 488 Low 30.5
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 482/678 B8 (669–687) 488 Low 12.8
PerCP-Cy5.5 482/695 B9 (688–707) 488 Medium 40.7
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
APC 650/660 R1 (652–669) 640 High 230.6
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
APC-R700 652/704 R4 (707–727) 640 High 167.1
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-Cy7 650/785 R7 (772–795) 640 Medium 219.0
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 Dye Products

NovaFluor dye - conjugated antibodies

We offer antibodies conjugated to many NovaFluor dyes to accommodate your flow cytometry needs.

 

NovaFluor CD4 Label Characterization Kits

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.

 

NovaFluor dye conjugation kits and blocking agents

Spectral Flow Cytometry Fundamentals

Learn about the history of spectral flow cytometry, the players in the marketplace, and the technological differences among spectral instrumentation. Whether you are a beginner or an advanced user, these fundamentals will help you conceptualize advances in spectral flow cytometry and how to think about designing experiments for a spectral cytometer.

For Research Use Only. Not for use in diagnostic procedures.