Advanced flow cytometry requires advanced tools. BioLegend’s Fire tandem fluorophores push the limits of your flow capabilities by expanding into spectral spaces previously unused in conventional cytometry. In addition, we also offer Fire dyes with enhanced stability and brightness properties for existing channels. Ignite your next discovery with these phycobiliprotein (PE, APC) and PerCP-based dyes, specifically designed for spectral cytometry and other advanced flow cytometry applications.

Spectral cytometry allows for the simultaneous use of fluorophores with spectral characteristics too similar to be distinguished from each other on conventional cytometers. PE/Fire™ 640 has a distinct emission peak between the peaks of PE/Dazzle™ 594 and PE/Cyanine5, which allows it to be accurately unmixed from these highly overlapping fluorophores. PE/Fire™ 640 was designed to be used for spectral cytometry, but may be used on conventional cytometers with filters optimized by the end user. PE/Fire™ 640 is recommended for: (1) labeling an antigen that is expressed on a small subset of cells in the sample, (2) labeling an antigen that will be excluded from final analysis, and (3) labeling an antigen that requires a bright signal but is not co-expressed with an antigen detected by PE/Cyanine5.

 

Excitation and Emission Spectra of PE/Fire™ 640

 

 

 

 

Emission spectra (top) and normalized emission spectra (middle) of PE/Fire™ 640 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare PE/Fire™ 640 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra (bottom) of PE/Fire™ 640 obtained from a spectrophotometer. To compare PE/Fire™ 640 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

 

Spectral Spillover of PE/Fire™ 640 

 

 

Spillover impact of PE/Fire™ 640 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.

 

 

Multicolor Compatibility of PE/Fire™ 640 

 

 

 

Multicolor Panel:

 

Marker

Clone

Fluorophore

CD4

SK3

PE/Fire™ 640

CD3

UCHT1

PE/Cyanine5

CD19

HIB19

PE/Dazzle™ 594

CD56

HCD56

BV605™

CD16

3G8

PE

CD8

RPA-T8

BV650™

 

 

RBC-lysed and washed human blood was stained with optimal test concentrations of the antibodies listed in the left panel. Blocking buffers were used as appropriate.

 

Titration Curve for PE/Fire™ 640

 

Titration curves for anti-CD4 PE/Dazzle™ 594 and anti-CD4 PE/Fire™ 640. Antibodies were titrated from 0-2 μg/test, and used to stain RBC-lysed human blood. Cells were analyzed on a Cytek® Aurora.

 

PE/Fire™ 640 Stability

 

Photostability

 


 

Two conditions of anti-CD4 PE/Fire™ 640 photostability were tested: (1) antibodies formulated at the optimal test concentration were left under fluorescent lighting or protected from light, then used to stain human PBMCs (indicated as “Ab only” in the graphs) and (2) antibodies were used to stain human PBMCs, which were then fixed and left in FluoroFix™ either under fluorescent lighting or protected from light (indicated as “Ab+cells” in the graphs). The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right).

 

 

Stability in Fixatives 

 

 

RBC-lysed human blood cells were stained with anti-CD4 PE/Fire™ 640 and fixed according to the recommended protocols for each fixative. Cells were analyzed directly after fixation and washing (fresh), or analyzed after being stored overnight in Cyto-Last™ Buffer. PE/Fire™ 640 is resistant to paraformaldehyde fixation, but is sensitive to organic solvent-containing phospho-specific fix and perm buffers (e.g. True-Phos™). The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). Learn more about our flow cytometry buffers.

 

Heat stability 

 

 

Antibodies were aliquoted into a sealed vial to avoid evaporation and stored at 4°C, 22°C, or 37°C for up to 28 days. The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). PE/Fire™ 640 does not have a significant reduction in staining index over 28 days at room temperature (22°C), but is reduced by 40% in staining index after 28 days at 37°C. There is no change in compensation, and the loss of staining index is due to an increase in non-specific binding of the negative population, not a loss of fluorescence intensity.

 

Note: Percent compensation for PE/Fire™ 640 heat stability was obtained on a conventional flow cytometer. Percent compensation for each other experiment was obtained on a 5L Cytek® Aurora by measuring percent spillover from the tandem dye into the donor fluor (PerCP, PE, or APC) peak emission channel. Percent spillover and compensation values on a conventional cytometer may differ significantly.

PE/Fire™ 700 ranks high on the brightness scale, comparable to PE/Cyanine5.5. Due to its significant spillover, PE/Fire™ 700 may be best suited for detecting antigens that are lowly and discreetly expressed. For example, the brightness of PE/Fire™ 700 can be leveraged to identify markers of rare populations that are not expressed on more abundant cell types. PE/Fire™ 700 is useful on spectral cytometers like the Cytek® Aurora and is also compatible with advanced conventional cytometers including the BD FACSymphony™. For both spectral and conventional cytometry, PE/Fire™ 700 can serve as a more stable alternative to PE/Cyanine5.5.

 

Excitation and Emission Spectra of PE/Fire™ 700

 

 

 

 

Emission spectra (top) and normalized emission spectra (middle) of PE/Fire™ 700 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare PE/Fire™ 700 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.


Normalized excitation and emission spectra (bottom) of PE/Fire™ 700 obtained from a spectrophotometer. To compare PE/Fire™ 700 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

 

Spectral Spillover of PE/Fire™ 700 

 

 

Spillover impact of PE/Fire™ 700 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.

 

 

Multicolor Compatibility of PE/Fire™ 700 

 

 

 

Multicolor Panel:

 

Marker

Clone

Fluorophore

CD4

SK3

PE/Fire™ 700

CD8

SK1

PerCP

CD19

HIB19

PE/Cyanine5

CD56

5.1H11

PE/Dazzle™ 594

CD154

24-31

PE/Fire™ 640

 

 

RBC-lysed and washed human blood was stained with a multicolor panel with an intentionally high degree of spectral complexity and overlap (left). Cells were analyzed on a 3-laser (top panels) or 5-laser Cytek® Aurora (bottom panels). Optimal test size concentrations were used for each antibody. Cells were gated on CD19-/CD56- events, and CD154 (CD40L) PE/Fire™ 640 is displayed against CD4 PE/Fire™ 700 or CD8 PerCP. This panel is able to be unmixed on both a 3-laser and a 5-laser Aurora. However, both the intensity of the PerCP signal and the spreading error of PE/Fire™ 640 is less substantial using a system that has both a 488 nm blue laser and a 561 nm yellow/green laser (Bottom panels). Spreading error only poses a problem when the brightness of the two fluors being plotted is not of sufficient intensity to resolve the two populations.

 

 

PE/Fire™ 700 Unmixing from PE/Cyanine5.5

 

 

RBC-lysed and washed human blood was co-stained with anti-CD19 PE/Fire™ 700 and either anti-CD4 PE/Cyanine5.5 (left) or a non-overlapping control fluor – in this case, anti-CD4 PE (right). Cells were acquired on a 5-laser Cytek® Aurora and gated on lymphocytes. When compared to the non-overlapping control (right), cells co-stained with PE/Fire™ 700 and PE/Cyanine5.5 (left) display a similar pattern of percent positive populations. This shows that it is possible to unmix PE/Fire™ 700 from PE/Cyanine5.5, but panel design should be carefully considered.

 

 

PE/Fire™ 700 and PE/Cyanine5.5 Brightness Comparison

 

RBC-lysed and washed human blood was stained with anti-CD4 PE/Fire™ 700 (red) or anti-CD4 PE/Cyanine5.5 (blue).

 

 

Titration Curve for PE/Fire™ 700

 

 

Titration of anti-CD4 PE/Fire™ 700 and anti-CD4 PE/Cyanine5 staining on RBC-lysed and washed human blood. Titrations were performed within the optimal range for each individual fluorophore. These two fluorophores are similar in brightness but both also exhibit significant spillover into neighboring emission channels. The staining index tends to decrease with greater concentrations of antibody used – this is due to an increase in non-specific binding.

 

 

PE/Fire™ 700 Stability

 

Photostability

 

Two conditions of anti-CD4 PE/Fire™ 700 and anti-CD4 PE/Cyanine5.5 photostability were tested: (left) antibodies formulated at the optimal test concentration were left under fluorescent lighting or protected from light, then used to stain human PBMCs and (right) antibodies were used to stain human PBMCs, which were then fixed and stored in FluoroFix™ either under fluorescent lighting or protected from light. The long-term stability of antibody conjugates can be predicted by its brightness and, in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel.

 

 

Stability in Fixatives 

 

RBC-lysed and washed human blood cells were stained with anti-CD4 PE/Fire™ 700 or anti-CD4 PE/Cyanine5.5 and fixed according to the recommended protocols for each fixative. Cells were analyzed directly after fixation and washing (fresh), or analyzed after being stored overnight in Cyto-Last™ Buffer. PE/Fire™ 700 is resistant to paraformaldehyde fixation but is sensitive to organic solvent-containing phospho-specific fix and perm buffers (e.g. True-Phos™). The long-term stability of antibody conjugates can be predicted by its brightness, and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel. Learn more about our flow cytometry flow cytometry buffers.

 

Heat stability 

 

 

Antibodies were aliquoted into a sealed vial to avoid evaporation and stored at 4°C, 22°C, or 37°C for up to 28 days. The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). PE/Fire™ 700 exhibits a reduction of 12% in staining index over 28 days at room temperature (22°C) and 36% reduction in staining index at 37°C. There is no change in compensation and the loss of staining index is due to an increase in non-specific binding of the negative population, not a loss of fluorescence intensity.

 

Note: Percent compensation for each experiment was obtained on a 5-laser Cytek® Aurora by measuring percent spillover from the tandem dye into the donor fluor (PerCP, PE, or APC) peak emission channel. Percent spillover and compensation values on a conventional cytometer may differ significantly.

APC/Fire™ 750 was BioLegend’s first Fire tandem dye, originally developed to provide a more temperature and photostable alternative to APC/Cy7. APC/Fire™ 750 also has lower compensation requirements than APC/Cy7 conjugates while maintaining an equal level of brightness. Additionally, APC/Fire™ 750 has minimal non-specific binding to monocytes, as has been observed with APC/Cy7. Lastly, APC/Fire™ 750 is equivalent to or brighter than APC-H7 in all conjugates tested.

 

Excitation and Emission Spectra of APC/Fire™ 750

 

 

APC Fire 750 Aurora Spectra

 

APC Fire 750 Spectra

 

Emission spectra (top) and normalized emission spectra (middle) of APC/Fire™ 750 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare APC/Fire™ 750 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra of APC/Fire™ 750 obtained from a spectrophotometer (bottom). To compare APC/Fire™ 750 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

 

Spectral Spillover of APC/Fire™ 750

 

Spectral Spillover of APC/Fire™ 750

Spillover impact of APC/Fire™ 750 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.

 

 

 

 

Spectral Compatibility with Zombie NIR™ in Spectral Flow Cytometry

 

Unlike comparable APC tandem dyes like APC/Cy7 and others, APC/Fire™ 750 has advantageous spectral properties that allow it to be unmixed from the fixable viability dye Zombie NIR™ in spectral applications. This gives the APC/Fire™ 750 and Zombie NIR™ combination special utility on spectral flow cytometers, allowing the user to conveniently slot both a viability probe and antibody stain into a narrow region of detection.

 

Normalized emission spectra of APC/Fire™ 750 and Zombie NIR™ as run on a 5-laser Cytek® Aurora Spectral Cytometer.

 

 

Multicolor Compatibility of APC/Fire™ 750

 

Multicolor Compatibility of APC/Fire™ 750PMA+Ionomycin (6 hours, in the presence of Monensin) stimulated human PBMCs were stained with anti-CD4 Alexa Fluor® 647 and then treated with Fixation Buffer followed by permeabilization with 1X Intracellular Staining Permeabilization Wash Buffer and stained with anti-IFN-γ APC/Fire™ 750 for 30 min.

 

Brightness Comparison

 

Brightness ComparisonHuman whole blood was stained for 20 min with anti-CD3 conjugates of APC/Fire™ 750, APC/Cy7, APC-H7, or APC-eFluor® 780 at each manufacturer's recommended optimal dilution, followed by RBC lysis and wash steps. Histograms were gated on lymphocyte populations based on forward and side scatter.

 

Low Background Binding to Monocytes 

 

Human whole blood was stained with anti-CD3 conjugates of APC/Fire™ 750 or APC/Cy7. Histograms were gated on monocyte populations.

 

APC/Fire™ 750 Stability

 

Human whole blood was stained for 20 min with anti-CD3 conjugates of APC/Fire™ 750, APC/Cy7, APC-H7, or APC-eFluor® 780 at each manufacturer's recommended optimal dilution, followed by RBC lysis and wash steps. Cells were then treated with either PBS control, 1% paraformaldehyde (PFA) in PBS, 4% PFA followed by 0.1% saponin, BioLegend's True-Nuclear™ Fix/Perm Buffer Set, BD's Transcription Factor Buffer Set, or eBioscience's FoxP3/Transcription Factor Staining Buffer Set prior to analysis. Lymphocyte populations were gated on based on optimal forward and side scatter.

 

Heat Stability 

 

Heat StabilityA vial of APC/Cy7 or APC/Fire™ 750 was stored in the dark at either 4°C or 37°C for 81 days. At certain intervals, an aliquot of reagent was used to stain Veri-Cells™, a lyophilized human PBMC product that effectively removes donor dependent variation in staining. Cells were stained for 15 min at room temp in cell staining buffer.

APC/Fire™ 810 expands the range of our spectral detection farther into the infrared than any of our previous fluorophores. On a 5-laser Cytek™ Aurora (UV/V/B/YG/R), APC/Fire™ 810 has very limited spectral spillover into any other channel except PE/Fire™ 810 (coming soon). This means that it can be assigned to any antigen with moderate to abundant expression, and can be used in combination with any other fluorophore. As such, it is very useful for identifying antigens expressed broadly in the cell sample. APC/Fire™ 810 is also very useful for detecting variably expressed markers, like markers of activation, since it emits so far outside of the autofluorescence range. These characteristics make APC/Fire™ 810 potentially very convenient to add into pre-existing panel designs with minimal adverse impact.

 

Excitation and Emission Spectra of APC/Fire™ 810

 

 

 

 

Emission spectra (top) and normalized emission spectra (middle) of APC/Fire™ 810 as run on a 5-laser Cytek® Aurora Spectral Cytometer. To compare APC/Fire™ 810 with other fluorophores on a spectral cytometer, use our Aurora Spectral Analyzer tool.

 

Normalized excitation and emission spectra (bottom) of APC/Fire™ 810 obtained from a spectrophotometer. To compare APC/Fire™ 810 with other fluorophores, use our Fluorescence Spectra Analyzer tool.

 

 

Spectral Spillover of APC/Fire™ 810 

 

 

Spillover impact of APC/Fire™ 810 into detection channels of a 5-laser Cytek® Aurora Spectral Cytometer.

 

 

Multicolor Compatibility of APC/Fire™ 810

 

APC/Fire™ 810 Unmixing 

 

 

Panel 1:

 

Marker

Fluorophore

CD4

BV510™

CD3

Alexa Fluor® 700

CD14

Alexa Fluor® 647

CD19

APC/Fire™ 750

CD27

Spark NIR™ 685

CD56

PE/Cy7

Panel 2:

 

Marker

Fluorophore

CD4

APC/Fire™ 810

CD3

Alexa Fluor® 700

CD14

Alexa Fluor® 647

CD19

APC/Fire™ 750

CD27

Spark NIR™ 685

CD56

PE/Cy7

 

 

A common way to test the ability of a fluorophore to be unmixed from other fluorophores with heavily overlapping emission spectra is to create parallel staining panels where the fluorophore of interest (in this case, anti-CD4 APC/Fire 810) is replaced with a control antibody conjugated to a fluorophore with non-overlapping spectra (in this case, anti-CD4 BV510). The accuracy of the unmixing controls, the pattern of the sample staining, and the percent positive populations are all indicators that APC/Fire 810 can be cleanly unmixed from all of the other fluorophores currently available for the red laser in this combination. Above are flow plots of RBC-lysed whole human blood cells stained with the two panels.

 

Intracellular Staining 

 

 

PMA/Ionomycin-stimulated human PBMC were stained with either anti-CD3 BV421™ and anti-IFN-γ APC/Fire™ 810 (left), or with anti-CD3 BV421™ and anti-T-bet APC/Fire™ 810 (right). APC/Fire™ 810 is useful for both intracellular and intranuclear targets. Be aware that when constructing a multicolor panel, APC/Fire™ 810 might exhibit some sensitivity to solvents or oxidation conditions like photobleaching.

 

 

Titration Curve for APC/Fire™ 810 

 

Titration curve of anti-CD4 APC/Fire 810 staining on human PBMC.

 

 

APC/Fire™ 810 Stability

 

Photostability

 

Two conditions of anti-CD4 APC/Fire™ 810 photostability were tested: (1) antibodies formulated at the optimal test concentration were left under fluorescent lighting or protected from light, then used to stain human PBMCs (indicated as “Ab only” in the graphs) and (2) antibodies were used to stain human PBMCs, which were then fixed and left in FluoroFix™ either under fluorescent lighting or protected from light (indicated as “Ab+cells” in the graphs). The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right).

 

 

Stability in Fixatives 

 

 

RBC-lysed human blood cells were stained with anti-CD4 APC/Fire™ 810 and fixed according to the recommended protocols for each fixative. Cells were analyzed directly after fixation and washing (fresh), or analyzed after being stored overnight in Cyto-Last™ Buffer. The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). Learn more about our flow cytometry buffers.

 

Heat Stability 

 

 

Antibodies were aliquoted into a sealed vial to avoid evaporation and stored at 4°C, 22°C, or 37°C for up to 30 days. The long-term stability of antibody conjugates can be predicted by its brightness (staining index, left) and in the case of PE, PerCP and APC tandems, the percent compensation into the donor channel (right). APC/Fire™ 810 has little reduction in staining index over 30 days at room temperature (22°C) and maintains 67% of the original staining index at 37°C after 30 days. The loss of staining index is due to an increase in non-specific binding of the negative population, not a loss of fluorescence intensity. There is a small change in compensation due to loss of acceptor fluors in the tandem.

 

 

Note: Percent compensation for each experiment was obtained on a 5L Cytek® Aurora by measuring percent spillover from the tandem dye into the donor fluor (PerCP, PE, or APC) peak emission channel. Percent spillover and compensation values on a conventional cytometer may differ significantly.

 

 

 

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