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Invitrogen NovaFluor dyes for immunophenotyping by flow cytometry
Add markers with minimal impact to compensation with NovaFluor dyes
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 are ideal additions to our portfolio of spectral antibodies, spectral dyes and spectral reagents.
Benefits to adding NovaFluor dyes include:
- Easy to add to existing panels, compatible in all sized panels for conventional and spectral flow cytometry
- Designed for minimal cross-laser excitation, minimal impact to compensation and decreased spillover spread for higher resolution
- Narrow excitation spectra enable more fluorophores per panel
- Unique spectral signatures for spectral flow cytometry
- Stable dyes that can be stored long-term without losing fluorescence as compared to tandem dyes
NovaFluor dye technology
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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 excitation/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.
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 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 into 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 12-color panel involving traditional dyes compared to a 14-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. Replacement with specific NovaFluor dyes removed cross-laser exciation , allowing for full use for blue and yellow laser.
Increase panel size by replacing conventional tandem dyes with unique NovaFluor dyes
| Set up (8-color panel) with conventional tandem dyes | Fluorophore | Antigen | Set up (9-color panel) with NovaFluor dyes | Fluororophore | Antigen |
| V1 | Live/Dead Violet | Viability | V1 | Live/Dead Violet | Viability |
| V2 | BV510 | CD16 | V2 | BV510 | CD16 |
| V3 | BV605 | CD25 | V3 | BV605 | CD25 |
| V4 | V4 | ||||
| B1 | FITC | CD3 | B1 | FITC | CD3 |
| B2 | B2 | NovaFluor Blue 610-30S | CD19 | ||
| B3 | B3 | ||||
| Y1 | Y1 | ||||
| Y2 | PE-eFluor 610 | CD4 | Y2 | NovaFluor Yellow 610 | CD4 |
| Y3 | Y3 | ||||
| Y4 | PE-Cy7 | CD69 | Y4 | PE-Cy7 | CD69 |
| R1 | AF647 | CD56 | R1 | AF647 | CD56 |
| R2 | R2 | ||||
| R3 | APC-eFluor 780 | CD8 | R3 | APC-eFluor 780 | CD8 |
Figure 4. Replacing conventional tandem dyes with NovaFluor dyes allows incorporation of additional markers in the panel. Normal human peripheral blood mononuclear cells (1 x 106 cells/well) were stained in 1x PBS using 1 µL of LIVE/DEAD Fixable Violet (Cat. No. L34955) for 30 minutes. Cells were washed and then blocked using CellBlox 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, top panel- using anti-human CD4 PE-eFluor 610 (5 µL, Cat. No. 61-0049-42) and the bottom-panel replacing PE-eFluor with anti-human CD4 NovaFluor Yellow 610 (5 µL, Cat. No. H001T02Y03) and anti-human CD19 NovaFluor Blue 610-70S (5 µL, Cat. No. H004T03B06). 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) overnight. Cells were then washed with Flow Cytometry Staining Buffer 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.
NovaFluor dye conjugated antibodies display lower spillover in comparison to the conventional tandem dye conjugated antibodies
Compensation errors due to spillover with traditional tandem dyes effectively corrected with the use of NovaFluor dyes
| 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 6. Use of NovaFluor dyes effectively correct 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 (Cat. No. L34955) for 30 minutes. Cells were washed and then blocked using CellBlox 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 anti-human CD4 PerCP-Cy5.5 (5 µL, Cat. No. 45-0049-42) and anti-human CD8 PE-Cy5.5 (5 µL, Cat. No. 35-0088-42), and the other replacing PerCp-Cy5.5 with anti-human CD4 NovaFluor Blue 660-120S (5 µL, Cat. No. H001T03B08) and replacing PE-Cy5.5 with anti-human CD8 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.
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) are able to clearly resolve cellular populations in our T cells panel, allowing for proper downstream analysis and expanded panel selection.
NovaFluor performance with Cytek Aurora (spectral)
Performance data of NovaFluor conjugates, studied with 5-laser Cytek Aurora under variety of staining conditions in spectral flow cytometry. NovaFluor dyes are engineered for spectral as well as conventional flow cytometry. NovaFluor dyes are compatible with Cytek Aurora spectral instruments, and the data below demonstrate that NovaFluor dye–conjugated antibodies show remarkable performance when exposed and tested under variety of different conditions.
Stable stain indices and MFI values for NovaFluor conjugates stored in fixatives, studied with 5-laser Cytek Aurora
A)
B)
| Type of NovaFluor dye | Time point | Stain index | MFI | Change in log MFI |
| NovaFluor Yellow 610 anti-human CD8 | Fresh | 28 | 10516 | |
| NovaFluor Yellow 610 anti-human CD8 | 4 hours | 28 | 9927 | -0.6% |
| NovaFluor Yellow 610 anti-human CD8 | 24 hours | 25 | 10306 | -0.2% |
| NovaFluor Yellow 610 anti-human CD8 | 5 days | 28 | 12054 | 1.5% |
| NovaFluor Blue 660-120S anti-human CD4 | Fresh | 181 | 37054 | |
| NovaFluor Blue 660-120S anti-human CD4 | 4 hours | 178 | 36267 | -0.2% |
| NovaFluor Blue 660-120S anti-human CD4 | 24 hours | 147 | 35715 | -0.4% |
| NovaFluor Blue 660-120S anti-human CD4 | 5 days | 170 | 34958 | -0.6% |
Figure 7. Stable stain indices and MFI values for NovaFluor dye conjugates stored in fixatives, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (2 x 106 cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01) and concurrently stained against Invitrogen NovaFluor Yellow 610 anti-human CD8 (10 µL; Cat. No. H003T02Y03), and Invitrogen NovaFluor Blue 660-120S anti-human CD4 (4 µL; Cat. No. H001T03B08) on ice, protected from light, for 30 minutes. Cells were fixed for 30 minutes using 50 µL of eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No.00-4222-26) and data was collected immediately afterward. Data from the same set of cells was collected again at 4-hours, 24-hours, and 5 days following staining. Cells were stored at 4°C, protected from light, between collection periods. All data was collected using a 5-laser Cytek Aurora using Cytek assay settings, at 30 µL/minute flow rate and 50,000 events were collected in the lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. (A) Time course stability of cells stained with NovaFluor dye–conjugated antibodies and stored in fixative. (B) Effect of fixation on stain indices and MFI of NovaFluor dye conjugates.
Stable similarity indices when NovaFluor conjugates are stored for up to 14 days in fixatives, studied with 5-laser Cytek Aurora
C)
| Similarity indices, stored in IC fixation buffer | ||
| Type of NovaFluor dye | 3 days | 14 days |
| NB510 | 1.00 | 1.00 |
| NB530 | 1.00 | 1.00 |
| NB555 | 1.00 | 1.00 |
| NB585 | 1.00 | 0.99 |
NB610-30S |
1.00 |
1.00 |
NB610-70S |
1.00 |
1.00 |
NB660-40S |
1.00 |
1.00 |
NB660-120S |
1.00 |
1.00 |
NY570 |
1.00 |
1.00 |
NY590 |
1.00 |
1.00 |
NY610 |
1.00 |
1.00 |
NY660 |
1.00 |
1.00 |
NY690 |
1.00 |
1.00 |
NY700 |
1.00 |
1.00 |
NY730 |
1.00 |
1.00 |
NR660 |
1.00 |
1.00 |
NR685 |
1.00 |
1.00 |
NR700 |
1.00 |
1.00 |
NR710 |
1.00 |
1.00 |
| Similarity indices, stored in Foxp3 fixation buffer | ||
| Type of NovaFluor dye | 3 days | 14 days |
NB510 |
1.00 |
1.00 |
NB530 |
1.00 |
1.00 |
NB555 |
1.00 |
0.97 |
NB585 |
1.00 |
0.99 |
NB610-30S |
1.00 |
1.00 |
NB610-70S |
1.00 |
0.98 |
NB660-40S |
1.00 |
1.00 |
NB660-120S |
1.00 |
0.99 |
NY570 |
1.00 |
1.00 |
NY590 |
1.00 |
1.00 |
NY610 |
1.00 |
0.99 |
NY660 |
1.00 |
1.00 |
NY690 |
1.00 |
1.00 |
NY700 |
1.00 |
1.00 |
NY730 |
1.00 |
1.00 |
NR660 |
1.00 |
1.00 |
NR685 |
1.00 |
1.00 |
NR700 |
1.00 |
1.00 |
NR710 |
1.00 |
0.99 |
Figure 8. Stable similarity indices when NovaFluor dye conjugates are stored for up to 14 days in fixatives, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01) and concurrently stained against Invitrogen NovaFluor Yellow 610 anti-human CD8 (10 µL; Cat. No. H003T02Y03), and Invitrogen NovaFluor Blue 660-120S anti-human CD4 (4 µL; Cat. No. H001T03B08) on ice, protected from light, for 30 minutes. Cells were fixed for 30 minutes using either 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26), or 25 µL eBioscience Fixation/Permeabilization Concentrate (Cat. No. 00-5123) combined with 75 µL eBioscience Perm Diluent (Cat. No. 00-5223) and data was collected immediately afterward. Data from the same set of cells was collected again at 3-days and 14-days following staining. Cells were stored at 4°C, protected from light, between collection periods. All data was collected using a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings and a 30 µL/minute flow rate. Briefly, similarity indices, a number between 0 and 1 that measures how closely a fluor’s spectral signature is to another fluor, were generated for each NovaFluor dye for freshly stained and fixed cells and compared to spectral signatures from cells that were stored for 3 and 14 days. If the similarity index was 0, the signatures were considered to have no similarity, and if the similarity index was ≥0.99, then the signatures were considered to be identical. This data demonstrates the fluorescent stability of NovaFluor conjugates stored in fixative. (A) Representative plots of NovaFluor conjugates stored in IC fixation Buffer (B) Representative plots of NovaFluor conjugates stored in Foxp3 fixation Buffer (C) Test results for similarity indices of cells stained with NovaFluor and stored in either Foxp3 Perm buffer or IC fixation buffer stored for 3 and 14 days.
NovaFluor conjugates are photostable upon prolonged exposure to light, studied with 5-laser Cytek Aurora
A)
B)
Type of NovaFluor dye |
4C vs. light exposed at room temperature |
4C vs. no light at room temperature |
NovaFluor Blue 510 |
-2.2% |
-0.8% |
NovaFluor Blue 530 |
-0.9% |
-0.3% |
NovaFluor Blue 555 |
0.0% |
0.3% |
NovaFluor Blue 585 |
-1.2% |
-4.1% |
NovaFluor Blue 610-30S |
-3.6% |
-2.7% |
NovaFluor Blue 610-70S |
-0.5% |
-3.8% |
NovaFluor Blue 660-40S |
-3.8% |
-2.5% |
NovaFluor Blue 660-120S |
-1.0% |
-3.7% |
NovaFluor Red 660 |
-1.9% |
-0.5% |
NovaFluor Red 685 |
-2.6% |
-1.6% |
NovaFluor Red 700 |
-2.7% |
-1.3% |
NovaFluor Red 710 |
-2.0% |
-0.3% |
NovaFluor Yellow 570 |
-1.0% |
-0.2% |
NovaFluor Yellow 590 |
-0.6% |
0.2% |
NovaFluor Yellow 610 |
-2.2% |
-1.8% |
NovaFluor Yellow 660 |
-4.6% |
-3.0% |
NovaFluor Yellow 690 |
-2.2% |
-1.6% |
NovaFluor Yellow 700 |
-3.1% |
-2.1% |
NovaFluor Yellow 730 |
-2.3% |
-0.7% |
Figure 9. NovaFluor dye conjugates are photostable upon prolonged exposure to light, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (2 x 106 cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01). Individual wells, in a 96 well plate, were concurrently single stained against anti-human CD4 using 4 µL/sample of NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, NovaFluor Yellow 570, NovaFluor Yellow 590, NovaFluor Yellow 610, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Red 660, NovaFluor Red 685, NovaFluor Red 700, and NovaFluor Red 710. Cells were stained on ice, protected from light, for 30 minutes. Cells were then fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26) and split into 3 equal parts. A set of cells were then protected from light and stored at 4°C, a set of cells were protected from light and stored at room temperature, and a set of cells was stored at room temperature and exposed to a 13W light for 18 hours. Following the 18-hour period, data from the 3 sets of cells was collected. All data was collected using a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings and a 30 µL/minute flow rate. 50,000 events were collected in the lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. Cells stored at 4°C were compared to both cells stored at room temperature protected from light and cells stored at room temperature exposed to light. Comparison was made by calculating the change in log median fluorescent intensity using the cells stored at 4°C as a baseline. A change of <±5% was considered acceptable. (A) Representative plots of NovaFluor conjugates when tested for photostability (B) Test results for change in log MFI was compared for cells stained with NovaFluor conjugates and tested for photostability.
Stable stain and separation indices of NovaFluor conjugates when stained and stored in a master mix containing CellBlox buffer, studied with 5-laser Cytek Aurora
A)
B)
| Type of NovaFluor dye | Time point | Stain index | MFI | Change in log MFI | Separation index |
NovaFluor Yellow 610 |
Fresh |
28 |
24 |
10366 |
|
NovaFluor Yellow 610 |
4 hours |
28 |
23 |
12195 |
1.7% |
NovaFluor Yellow 610 |
24 hours |
22 |
17 |
10129 |
-0.3% |
NovaFluor Yellow 610 |
5 days |
30 |
26 |
11606 |
1.2% |
NovaFluor Yellow 610 |
14 days |
22 |
26 |
11776 |
1.4% |
NovaFluor Yellow 610 |
30 days |
25 |
20 |
10983 |
0.6% |
NovaFluor Blue 660-120S |
Fresh |
178 |
178 |
36602 |
|
NovaFluor Blue 660-120S |
4 hours |
188 |
188 |
38681 |
0.5% |
NovaFluor Blue 660-120S |
24 hours |
197 |
194 |
41900 |
1.3% |
NovaFluor Blue 660-120S |
5 days |
228 |
224 |
44976 |
1.9% |
NovaFluor Blue 660-120S |
14 days |
202 |
198 |
39888 |
0.8% |
NovaFluor Blue 660-120S |
30 days |
206 |
194 |
52486 |
3.4% |
Figure 10. Stable stain and separation indices of NovaFluor dye conjugates when stained and stored in a master mix containing CellBlox buffer, studied with 5-laser Cytek Aurora. An antibody master mix consisting of Invitrogen NovaFluor Yellow 610 anti-CD8 (30 µL; Cat. No. H003T02Y03), Invitrogen NovaFluor Blue 660-120S anti-CD4 (12 µL; Cat. No. H001T03B08),Invitrogen CellBlox Blocking Buffer (528 µL; Cat. No. B001T03F01), and eBioscience Flow Cytometry Staining Buffer (30 µL; Cat. No. 00-4222-26) was used to stain normal human peripheral blood mononuclear cells (1 x 106 cells/sample). Antibody conjugates and CellBlox buffer were used at manufacturer suggested concentrations, and flow cytometry staining buffer was used to achieve a final staining volume of 100 µL. Cells were stained on ice, protected from light for 30 minutes, immediately, 4 hours, 24 hours, 5 days, 14 days, and 30 days following formation of the master mix. The antibody master mix was stored at 4°C, protected from light. All cells were from the same donor. All cells were fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). Data was collected by a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings using a 30 µL/minute flow rate and 50,000 events were collected in they lymphocyte scatter gate. Data was unmixed using autofluorescence extraction included in the unmixing algorithm. (A) Representative plots of NovaFluor conjugates when tested for performance in CellBlox buffer for up to 30 days (B) Test results for stain indices, separation indices, MFI and change in log MFI for cells stained and stored with a mastermix containing NovaFluor conjugates and CellBlox buffer for up to 30 days.
Comparable similarity indices of NovaFluor dye conjugates with Ultracomp eBeads and UltraComp eBeads Plus in comparison to fresh cells, studied with 5-laser Cytek Aurora
A)
B)
| Similarity indices in comparison to cells | ||
| Type of NovaFluor dye | UltraComp | UltraComp Plus |
NovaFluor Blue 510 |
1.00 |
1.00 |
NovaFluor Blue 530 |
1.00 |
0.99 |
NovaFluor Blue 555 |
0.99 |
1.00 |
NovaFluor Blue 585 |
0.99 |
1.00 |
NovaFluor Blue 610-30S |
0.99 |
0.99 |
NovaFluor Blue 610-70S |
1.00 |
1.00 |
NovaFluor Blue 660-40S |
1.00 |
1.00 |
NovaFluor Blue 660-120S |
1.00 |
1.00 |
NovaFluor Yellow 570 |
1.00 |
1.00 |
NovaFluor Yellow 590 |
1.00 |
1.00 |
NovaFluor Yellow 610 |
1.00 |
1.00 |
NovaFluor Yellow 660 |
1.00 |
1.00 |
NovaFluor Yellow 690 |
1.00 |
1.00 |
NovaFluor Yellow 700 |
1.00 |
1.00 |
NovaFluor Yellow 730 |
0.99 |
0.99 |
NovaFluor Red 660 |
1.00 |
1.00 |
NovaFluor Red 685 |
1.00 |
1.00 |
NovaFluor Red 700 |
1.00 |
1.00 |
NovaFluor Red 710 |
1.00 |
1.00 |
Figure 11. Comparable similarity indices of NovaFluor dye conjugates with UltraComp eBeads and UltraComp eBeads Plus in comparison to fresh cells, studied with 5-laser Cytek Aurora. Normal human peripheral blood mononuclear cells (1 x 106 cells/well) were blocked using 5 µL of Invitrogen CellBlox Blocking Buffer (Cat. No. B001T03F01). Individual wells, in a 96 well plate, were concurrently single stained against anti-human CD4 using 2 µL/sample of NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610-30S, NovaFluor Blue 610-70S, NovaFluor Blue 660-40S, NovaFluor Blue 660-120S, NovaFluor Yellow 570, NovaFluor Yellow 590, NovaFluor Yellow 610, NovaFluor Yellow 660, NovaFluor Yellow 690, NovaFluor Yellow 700, NovaFluor Yellow 730, NovaFluor Red 660, NovaFluor Red 685, NovaFluor Red 700, and NovaFluor Red 710. Cells were stained on ice, protected from light, for 30 minutes. Cells were then fixed for 30 minutes using 50 µL eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) combined with 50 µL of eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26). The same fluors were used to stain UltraComp eBeads (Cat. No. 01-2222-42) and UltraComp eBeads Plus (Cat. No. 01-3333-42) at the rate of 1 µL/test for 15 minutes, protected from light, on ice. All data was collected using a 5-laser Cytek Aurora with the manufacturer's recommended Cytek assay settings and a 30 µL/minute flow rate. 50,000 events were collected in the lymphocyte scatter gate for cells and 5,000 events were collected for each set of beads. Briefly, similarity indices, a number between 0 and 1 that measures how closely a fluor’s spectral signature is to another fluor, were generated for each NovaFluor dye for freshly stained and fixed cells and compared to spectral signatures from UltraComp and UltraComp eBeads Plus. If the similarity index was 0, the signatures were considered to have no similarity, and if the similarity index was ≥0.99, then the signatures were considered to be identical. This data demonstrates that UltraComp and UltraComp eBeads Plus can be used to unmix cells stained with NovaFluor dyes. (A) Representative plots of NovaFluor conjugates when tested for performance with human peripheral blood mononuclear cells, UltraComp and UltraComp eBeads Plus (B) Test results for separation indices for cells, UltraComp or UltraComp eBeads Plus stained and stored with a mastermix containing NovaFluor conjugates.
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 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 |
| NovaFluor Blue 690 | 494/690 | B9 (688–707) | 488 | Low | 67.5 |
| 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 | NovaFluor Yellow 590 | 552/590 | YG2 (588–608) | 488; 561 | High |
| PE-Dazzle 594 | 566/610 | YG3 (605–625) | 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 |
| NovaFluor Yellow 755 | 552/755 | YG8 (735-765) | 561 | Low | 93 |
| 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 | NovaFluor Red 725 | 636/725 | R5 (728-749) | 637 | Medium | 116 | NovaFluor Red 755 | 636/755 | R6 (749-772) | 637 | Low | 118 |
| 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.
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 12).
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 13.
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 along with antibodies, 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.
Imagine spectral possibilities
Video expresses how much more information you can get from a cell using high parameter/spectral flow cytometry.
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.
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.
Related products and offers
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 NovaFluor dyes as direct conjugates of CD4. These kits can be used to evaluate performance of NovaFluor dyes on flow cytometers.
NovaFluor dye conjugation kits and blocking agents
For Research Use Only. Not for use in diagnostic procedures.