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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 by blocking dye-to-cell interactions, use Invitrogen CellBlox Plus Blocking Buffer.
Use the table below to select a NovaFluor dye and see available conjugated antibodies. Check stain index for brightness here
Ultra Violet |
Violet |
Blue |
Yellow |
Red |
(358/763 nm) |
(408/691 nm) |
(496/511 nm) |
(552/568 nm) |
(637/659 nm) |
(391/743 nm) |
(509/530 nm) |
(552/590 nm) |
(637/685 nm) |
|
(408/800 nm) |
(494/555 nm) |
(552/612 nm) |
(639/700 nm) |
|
(494/585nm) |
(552/663 nm) |
(639/710 nm) |
||
(509/614 nm) |
(552/690 nm) |
(636/725 nm) |
||
(509/614 nm) |
(552/700 nm) |
(636/755 nm) |
||
(509/665 nm) |
(552/731 nm) |
|||
(509/665 nm) |
(552/755 nm) |
|||
(494/690 nm) |
(551/813 nm) |
|||
(492/725 nm) |
||||
(490/764 nm) |
||||
(493/806 nm) |
||||
*This NovaFluor dye fits in a spectral space that is currently not accessible by other spectral dyes. |
Have an antibody that isn’t conjugated to a NovaFluor? Use our NovaFluor conjugation kits to create NovaFluor conjugates on-demand.
For more ultra violet and violet dye options explore Brilliant Ultra Violet™/Violet™ Polymer Dyes and Super Bright Polymer dyes.
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.
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.
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.
Invitrogen CellBlox Plus Blocking Buffer is formulated for enhanced blocking of nonspecific binding of NovaFluor antibody conjugates to cells. 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.
Discover the latest updates, data insights, and educational content from NovaFlour. Stay informed, explore new experiments, and enhance your scientific journey with our helpful tips and stories.
This blog delves into how spectrally cleaner NovaFluor dyes mitigate spillover effects, enabling the detection of rare and dim antigens with unprecedented clarity in multi-panel experiments.
For Research Use Only. Not for use in diagnostic procedures.