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Are brighter dyes better for flow cytometry?
Brighter fluorophores do tend to make rare antigens easier to observe if they increase the separation between the positive and negative populations without increasing the background on the off-target cells. They also work great if there is only a single target (i.e., one laser, one detector, one antigen) and the positive population’s Mean Fluorescence Intensity (MFI) is still within the detector’s dynamic range (i.e., not off scale). If two or more fluorophores are combined in a panel, however, the benefits of a brighter fluorophore become a little more complicated. The fluorescent properties of each dye-antibody conjugate must be taken into consideration to understand how they will impact each other. Many fluorophores emit light in multiple detectors and/or are cross-excited by multiple lasers.
What are the consequences of using the brightest fluorophores?
Plotting the absorbance and emission spectra of fluorophores can help predict how their spectral properties will impact a panel. The images in Figure 1 show an example spectra for PE-eFluor 610. While this tandem dye is primarily excited by the yellow laser line, it is also cross-excited quite heavily by the blue laser. Similarly, this dye emits well in its primary detector (YL-2), but it also has significant spillover into other channels, like YL-1. Thus, while PE-eFluor 610 is very bright and can be well-suited for resolving dim populations, its spillover and cross-excitation have the potential to reduce the resolution in other detectors when used in a panel.
Figure 1. Both images were adapted from the Fluorescence SpectraViewer using the stock configuration of the Attune NxT Flow Cytometer, blue/red/violet/yellow (V4). PE-eFluor 610’s absorbance spectrum is overlaid with the standard violet (405 nm), blue (488 nm), yellow (561 nm), and red (640 nm) laser lines and arrows indicate the yellow laser as the primary excitation source and the blue laser as the secondary excitation source. PE-eFluor 610’s emission spectrum from the primary excitation source highlights the primary detector, YL-2 (620/15) as well as the secondary detector, YL-1 (585/16) (right). Note that there is significant spillover into the secondary detector, YL-1, for PE-eFluor 610.
Although using the brightest fluorophore possible for every antigen in a panel seems like a wonderful idea, there may be problematic consequences. The sample data in Figure 2 shows an example where brightness is not always better. Using increasing antigen density and a bright fluorophore (in this case PE), brightness increases in the primary detector (YL-1) and the spread into secondary detectors also increases (seen in red, pink, and purple). The increased spread of the brighter conjugates results in difficulties identifying co-stained populations. If the brightness of the PE conjugate is reduced, however, the separation can become more usable.
Figure 2. Normal human peripheral blood cells were treated with Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-73) for 15 minutes at 4°C and then stained using the indicated antibody cocktails. Antibody cocktails included CellBlox Blocking Buffer. We recommend using CellBlox Plus Blocking Buffer for improved results (Cat. No. C001T06F01). Cells in the lymphocyte gate were used for analysis. For the purposes of illustrating increasing spread, CD8a was gated on the brightest expressing cells (CD8a hi). Data was collected using the stock configuration of the Attune NxT Flow Cytometer, blue/red/violet/yellow (V4). Single stained controls of PE (primary detector YL-1) compensated into the BL-2 detector, in order of increasing antigen density: Unstained (black), CD197 (CCR7) Monoclonal Antibody (3D12), PE (Cat. No. 12-1979-42) (purple), CD27 Monoclonal Antibody (O323), PE (Cat. No. 12-0279-42) (pink), CD8a Monoclonal Antibody (SK1), PE (Cat. No. 12-0087-42) (red). MFI (i.e., brightness) of YL-2 detector and rSD (i.e., spread) of the BL-2 detector reported next to each population (left). Single stained controls of CD197 (CCR7) PE (purple), CD27 PE (pink), CD8 PE (red) overlayed with matched co-stained samples that include CD62L (L-Selectin) Monoclonal Antibody (DREG-56), NovaFluor Blue 585 (Cat. No. H009T03B04) (black) (right).
How can NovaFluor dyes help?
The best way to overcome a lack of resolution due to spread is to build panels using spectrally clean fluorophores. NovaFluor dyes are engineered to reduce cross-excitation and spillover into off-target channels. In Figure 3, PE-eFluor 610 is compared to a spectrally cleaner fluorophore substitution, NovaFluor Yellow 610. Although both fluorophores occupy the same primary detector on the Attune, NovaFluor Yellow 610 has nearly a 65% decrease in normalized cross-excitation by the blue laser, which results in less emission into BL-2, the secondary detector most impacted by PE-eFluor 610. Considering that PE-eFluor 610 is a brighter dye, the impact of this cross-excitation is even greater.
Figure 3. Both images were adapted from the Fluorescence SpectraViewer using the stock configuration of the Attune NxT Flow Cytometer, blue/red/violet/yellow (V4). Absorbance spectra of PE-eFluor 610 (yellow) and NovaFluor Yellow 610 (orange) overlayed with the blue (488 nm) and yellow (561 nm) laser lines (left). Emission profiles of the same two fluorophores due to cross-excitation of the blue laser (488 nm) are displayed. Both spectra were scaled by their primary excitation source, yellow 561 nm. The yellow box indicates the BL-2 detector (590/40) (right).
Replacing the brighter PE-eFluor 610 with the dimmer and cleaner NovaFluor Yellow 610 should, in theory, result in less spillover. To test this in practice, each fluorophore was conjugated to the same antibody and used to stain the same cells from the same donor. In Figure 4, NovaFluor Yellow 610 has significantly less spillover and spread than PE-eFluor 610, and although it is dimmer, the NovaFluor Yellow 610 positive population can still be easily identified.
Figure 4. Normal human peripheral blood cells were treated with Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-73) for 15 minutes at 4°C and then stained using the indicated antibody cocktails. Antibody cocktails included CellBlox Blocking Buffer. We recommend using CellBlox Plus Blocking Buffer for improved results (Cat. No. C001T06F01). Cells in the lymphocyte gate were used for analysis. Data was generated using the stock configuration of the Attune NxT Flow Cytometer, blue/red/violet/yellow (V4). Single stained controls of (A) uncompensated and (B) compensated CD4 Monoclonal Antibody (SK3), PE-eFluor 610 (Cat. No. 61-0047-42) (left – red) and CD4 Monoclonal Antibody (SK3), NovaFluor Yellow 610 (Cat. No. H001T03Y03) (right – blue) on the YL-2 (Primary) and BL-2 (Secondary) detectors. Positive population (indicated by the rectangle gates) percentages are reported in the upper right corner.
Historically, brighter fluorophores have been praised for the ability to detect dim populations. Several of those legacy fluorophores, however, have a large degree of spillover and spread due to off-target fluorescence. The spillover-spreading that is introduced decreases the resolution of panels, making it difficult to observe rare or dimly expressed antigens, particularly for co-expressed markers. Using spectrally cleaner conjugates results in less spillover-spreading, which allows the building of larger panels without sacrificing resolution.
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