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 1. Zellanalyse
 2. Flow Cytometry
 3. NovaFluor Dyes for Immunophenotyping


NOVAFLUOR DYES FOR IMMUNOPHENOTYPING

See your cells in a whole new light
Find NovaFluor antibodies

Flow Cytometry


TABLE OF CONTENTS

NovaFluor Dye Selection Guide
Minimalized Cross-Laser Excitation
Minimalized Impact to Compensation
Decreased Spillover Spread
Designed for Panel Expansion
Invitrogen CellBlox Plus Blocking Buffer
Related Products
Resources
Learn more about our NovaFluor sample program for your lab

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:

 * Minimalized cross-laser excitation
 * Minimalized impact to compensation
 * Decreased spillover spread
 * Designed for panel expansion

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.


SEE MINIMAL CROSS-LASER EXCITATION

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.                                                          
                                                                               
                  

Top


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.


SEE 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.

Top


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.


SEE DECREASED SPILLOVER

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.

Top


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.


SEE HOW TO EXPAND PANELS

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).

Top


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.


SEE BENEFITS OF CELLBLOX PLUS BLOCKING BUFFER

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.

Top


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.

Learn more



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.

Learn more



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.

Learn more



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.

Learn more


--------------------------------------------------------------------------------


RESOURCES


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

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

Learn more


--------------------------------------------------------------------------------

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

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