NovaFluor Dyes for Immunophenotyping

See your cells in a whole new light

NovaFluor dyes are designed with narrow excitation for minimal cross-laser excitation for spectral and traditional flow cytometry to deliver high resolution. 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. NovaFluor conjugated antibodies represent foundational technology in the rapidly expanding world of flow cytometry. Learn more about our total spectral flow cytometry offerings.

Explore more about the advantages of using NovaFluor dyes:

And to enhance NovaFluor data resolution with minimal changes to most flow cytometry staining protocols use Invitrogen CellBlox Plus Blocking Buffer.


NovaFluor dye selection guide

Use the table below to select a NovaFluor dye and see available conjugated antibodies.

Ultra Violet

Violet

Blue

Ex/Em max (nm)

Yellow

Ex/Em max (nm)

Red

Ex/Em max (nm)

Explore our Brilliant Ultra Violet/Violet Polymer Dyes and Super Bright Polymer Dyes

NovaFluor Blue 510

496/511

NovaFluor Yellow 570

552/568

NovaFluor Red 660

637/659

NovaFluor Blue 530

509/530

NovaFluor Yellow 590

552/590

NovaFluor Red 685

637/685

NovaFluor Blue 555

494/555

NovaFluor Yellow 610

552/612

NovaFluor Red 700

639/700

NovaFluor Blue 585

494/585

NovaFluor Yellow 660

552/663

NovaFluor Red 710

639/710

NovaFluor Blue 610-30S

509/614

NovaFluor Yellow 690

552/690

NovaFluor Red 725

636/725

NovaFluor Blue 610-70S

509/614

NovaFluor Yellow 700

552/700

NovaFluor Red 755

636/755

NovaFluor Blue 660-40S

509/665

NovaFluor Yellow 730

552/731

   

NovaFluor Blue 660-120S

509/665

NovaFluor Yellow 755

552/755

   

NovaFluor Blue 690

494/690

       

NovaFluor Blue 725

492/725

       

NovaFluor Blue 760

490/764

       

Have an antibody that isn’t conjugated to a NovaFluor? Use our NovaFluor conjugation kits to create NovaFluor conjugates on-demand


Minimalized cross-laser excitation

Cross-laser excitation is a significant contributor to spillover in flow cytometry experiments. Some fluorophores have excitation curves that enable fluorescence on more than one laser, leading to excess spillover and consequent spreading error. These effects can reduce the maximum experiment complexity possible on any instrument. NovaFluor dyes are designed to minimize cross-laser excitation, thereby enabling the full potential of your flow cytometer.

A closer look at the excitation curve for PE. PE is well-excited by blue (488 nm), green (532 nm), and yellow-green (561 nm) laser excitation sources. This results in the fluorophore having cross-laser excitation. NovaFluor dyes have much narrower excitation curves optimized for the blue and yellow-green lasers most common on flow cytometers. Using NovaFluor dyes resolves the cross-laser excitation issues.

 

Figure 1. Excitation ranges of NovaFluor dyes compared to PE/Dazzle 594 dye. Excitation spectrum of PE/Dazzle 594 (left) and normalized excitation spectra of PE/Dazzle 594, NovaFluor Blue 610, and NovaFluor Yellow 610 (right) with blue (488 nm), green (532 nm), yellow (561 nm), and red (635 nm) laser lines overlaid. While PE/Dazzle 594 is well-excited by blue, green and yellow lasers, 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.                                                                                                                                                             

Minimalized impact to compensation

Increasing panel complexity can lead to compensation-related background. NovaFluor dyes are designed to minimize impacts to compensation, making it easier to increase panel size and complexity with minimal impact to compensation.

Typically, researchers do not combine PerCP-Cy5.5 and PE-Cy5.5 in the same panel due to spillover and compensation errors. Data here demonstrate compensation errors observed when using the tandem dyes PerCP-Cy5.5 and PE-Cy5.5 together in a panel collected on a default Attune cytometer setup (top row). Spillover from PerCp-Cy5.5 into the YL3 channel results in spreading of the PerCP-Cy5.5 negative populations, resulting in populations that do not resolve properly. Incorrect compensation can also lead to downstream analysis errors, as shown in the CD45RA+CD45RO+ population (top row) that does not exist when the CD4 and CD8 populations are separated properly (bottom row) using NovaFluor dyes. NovaFluor dyes that occupy the same primary channels as PerCp-Cy5.5 and PE-Cy5.5 (BL3 and YL3) can clearly resolve cellular populations in our T cells panel, allowing for proper downstream analysis and expanded panel selection.

Set up (5-color panel) with conventional tandem dyes

Fluorophore

Antigen

Set up (6-color panel) with NovaFluor dyes

Fluorophore

Antigen

V1

Live/Dead Violet

Viability

V1

Live/Dead Violet

Viability

V2

 

 

V2

 

 

V3

 

 

V3

 

 

V4

 

 

V4

 

 

B1

FITC

CD3

B1

FITC

CD3

B2

 

 

B2

 

 

B3

PerCp Cy5.5

CD4

B3

NovaFluor Blue 660-120S 

CD4

Y1

PE

CD45RA

Y1

PE 

CD45RA

Y2

 

 

Y2

 

 

Y3

PE Cy5.5

CD8

Y3

NovaFluor Yellow 660

CD8

Y4

 

 

Y4

 

 

R1

 

 

R1

 

 

R2

 

 

R2

 

 

R3

APC-eFluor 780

CD45RO

R3

APC-eFluor 780

CD45RO

Figure 2. Use of NovaFluor dyes effectively corrects the compensation errors seen when using conventional fluorophores. Normal human peripheral blood mononuclear cells (1 x 106 cells/well) were stained in 1x PBS using 1 µL of LIVE/DEAD Fixable Violet Dead Cell Stain Kit for 405nm excitation (Cat. No. L34955) for 30 minutes. Cells were washed and then blocked using CellBlox Blocking Buffer (Cat. No. B001T03F01) and Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-71) in eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). Cells were then stained in two separate panels, one using CD4 Monoclonal Antibody (RPA-T4), PerCP-Cyanine5.5 (5 µL, Cat. No. 45-0049-42) and CD8a Monoclonal Antibody (RPA-T8), PE-Cyanine5.5 (5 µL, Cat. No. 35-0088-42), and the other replacing PerCp-Cy5.5 with CD4 Monoclonal Antibody (SK3 (SK-3)) NovaFluor Blue 660-120S (5 µL, Cat. No. H001T03B08) and replacing PE-Cy5.5 with CD8a Monoclonal Antibody (OKT8 (OKT-8)), NovaFluor Yellow 660 (5 µL, Cat. No. H003T03Y04). Cells were stained for 30 minutes at 4°C, washed, and then fixed using 100 µL of eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) for 30 minutes. Cells were then washed, and data was collected on an Attune CytPix Flow Cytometer (4-laser configuration) from 30,000 cells from the lymphocyte gate. Collected data was compensated and later analyzed using FlowJo® software.

Decreased spillover spread

Spillover spread is impacted by several factors including fluorophore emission-spectra overlap, antigen expression level, and cross-laser excitation. Fluorophores with high cross-laser excitation can increase spread to a level where a detector will no longer be usable due to excessive background.

Fluorophores designed to avoid cross-laser excitation can be valuable in the expansion of usable detectors on a flow cytometry instrument. NovaFluor dye conjugated antibodies display lower cross-laser spillover in comparison to the conventional PE tandem dye conjugated antibodies.

Figure 3. Spillover reduction using NovaFluor antibodies to replace conventional tandem dyes. UltraComp eBeads Plus Compensation Beads (Cat. No. 01-3333-42) were single stained with either CD4 Monoclonal Antibody (RPA-T4), PE-eFluor 610 (5 µL, Cat. No. 61-0049-42), CD4 Monoclonal Antibody (SK3 (SK-3)) NovaFluor Yellow 610  (5 µL, Cat. No. H001T02Y03), or CD19 Monoclonal Antibody (HIB19), NovaFluor Blue 610-70S (5 µL, Cat. No. H004T03B06). Beads were stained for 30 minutes in eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26), washed, and then analyzed on an Attune CytPix Flow Cytometer (4-laser configuration) (red arrows show spillover). Data shown here are raw fluorescent signal in the BL2 and YL2 channels for each fluorophore. Signal intensity is shown here (x axes) in the YL2 and BL2 channels, demonstrating the strong spillover in BL2 from PE-eFluor 610 dye. This spillover is mitigated with the use of NovaFluor dyes that occupy the same primary channels (BL2, YL2), allowing correct compensation and increased panel size.

Designed for panel expansion

NovaFluor dyes’ enhanced spectral characteristics make them excellent candidates for panel expansion with minimal impacts to spillover and consequent spreading error.

Unlock the full power of your instrument through the enhanced spectral properties of NovaFluor dyes. Narrow excitation spectra enable broader utilization of the blue and yellow-green lasers on many flow cytometers. In this example, we see the typical channel distribution of a 12-color panel involving traditional dyes compared to a 14-color panel achieved by incorporating NovaFluor dyes.

Table 1. Design of two multicolor flow cytometry panels showing panel expansion with NovaFluor dyes. Panel A is 12-conjugate panel using traditional conjugates and Panel B is a 14-conjugate panel employing NovaFluor conjugates. Top rows indicate detectors and filter bandwidths, which are grouped by excitation source in order of increasing wavelength. Surface antigens are reported with conjugated fluorophores indicated. Exchanges in fluorophores are highlighted by color, while unoccupied detectors are indicated by “Open”. For example, CD3 PerCP-Cyanine5.5 used in Panel 1 is replaced by CD3 NovaFluor Blue 610-70S in Panel 2.
 

Figure 4. Data from two multicolor flow cytometry panels showing panel expansion with NovaFluor dyes. Comparison of flow cytometry plots as shown in Table 1 with the 12-conjugate panel (A) and 14-conjugate panel (B). All plots were gated on singlet, then viability, and finally lymphocytes. In addition, specific plots were sub-gated on populations as indicated above. Note arrows indicating areas of increased spread for Panel 1 vs Panel 2. Human peripheral blood mononuclear cells were stained Fixable Viability Dye eFluor 506 (Cat. No. 65-0866-18) then blocked with Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-73) and finally stained with the antibody conjugates indicated in Table 1 in the presence of CellBlox Blocking Buffer (Cat. No. B001T06F01) and Brilliant Stain Buffer (Cat. No. 00-4409-75).

Invitrogen CellBlox Plus Blocking Buffer

Invitrogen CellBlox Plus Blocking Buffer is formulated for enhanced blocking of nonspecific binding of NovaFluor antibody conjugates. It provides exceptional blocking of nonspecific interactions when compared to CellBlox Blocking Buffer. Use of CellBlox Blocking Plus Buffer requires minimal change to most flow cytometry staining protocols.

Always use CellBlox Blocking Plus Buffer with NovaFluor dyes when labeling cells for best background reduction

 

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

Figure 5. CellBlox Plus Blocking buffer offers improved blocking compared to CellBlox Buffer. Peripheral blood mononuclear cells (PBMCs) were either unblocked (A), blocked with CellBlox Blocking Buffer (B001T06F01) (B), or CellBlox Plus Blocking Buffer (C001T06F01) (C). Cells were then stained with either CD4 monoclonal antibody, NovaFluor Yellow 755 (top) or CD4 monoclonal antibody, NovaFluor Red 710 (bottom). All cells were co-stained with CD19 Monoclonal Antibody (HIB19), eFluor 450 (48-0199-42). Two-dimensional pseudocolor plots of CD4 vs CD19 show that the CellBlox Plus Blocking Buffer reduces non-specific binding compared to staining without CellBlox Plus Blocking Buffer and with CellBlox Blocking Buffer. Data were acquired on a 5-laser Cytek Aurora Flow Cytometer.

 


Figure 6. CD3 labeling, combined with CellBlox Plus Blocking Buffer, is shown to reduce background in lymphocytes and non-specific labeling of monocytes and macrophages compared with labeling without CellBlox Plus Blocking Buffer.
Peripheral blood mononuclear cells (PBMCs) were either left unstained (black), stained with CD3 Monoclonal Antibody (UCHT1), NovaFluor Blue 660 120S conjugate (H002T03B08) with (red), and without (blue) the addition of CellBlox Plus Blocking Buffer (C001T06F01). (A) Forward Scatter vs. Side Scatter pseudocolor plot shows lymphocyte and monocyte gates. Histogram overlay plots show the expression of CD3 within the (B) lymphocyte and (C) monocyte gates. Data were acquired in the B7 channel on a 5-laser Cytek Aurora.

Related products

NovaFluor Conjugation Kits

Do you have an antibody that isn’t conjugated to a NovaFluor? Use our NovaFluor conjugation kits to create NovaFluor conjugates on-demand.

NovaFluor CD4 Label Characterization Kits

NovaFluor CD4 label characterization kits are formulated to enable the testing of NovaFluor dyes as direct conjugates of CD4. They can be used to evaluate performance of NovaFluor dyes on flow cytometers.

CellBlox Plus Blocking Buffer

CellBlox Plus blocking buffer is formulated to block nonspecific binding of NovaFluor dyes. It is a non-antibody blocking solution and should be utilized with NovaFluor dyes for labeling of any cell type.

Image displaying LIVE/DEAD Fixable Viability Dyes.

LIVE/DEAD fixable viability dyes are fixable viability dyes that help ensure accurate assessment of cell viability in samples after fixation and/or permeabilization.


Resources

FREE—Flow Cytometry Panel Design Service and Panel Builder Tool

Our scientists can design panels for you as many times you need—free of charge, no product purchase needed.

Fluorescence SpectraViewer

Assess the spectral compatibility of dyes and probes for experiments that utilize fluorescence detection techniques.