Bright fluorescent polymer dyes for the violet laser

Flow cytometry is an important technique that is central to many of the fastest-growing areas of life science research, including immuno-oncology, antibody therapeutic development, and gene editing. Not only does it enable simultaneous multiparametric analysis of proteins, gene expression, and cell functions at the single-cell level, but it also allows the collection of a statistically relevant amount of data from a heterogeneous cell population. In any efficient flow cytometry workflow, the instrumentation and reagents must work together to ensure reproducible, high-quality data. Here we describe the Invitrogen eBioscience Super Bright antibody conjugates, optimized for use in flow cytometry and designed to provide exceptionally bright readouts for better discrimination of cell subpopulations (Figures 1 and 2).

Super Bright Fluorescence

Figure 1. Emission spectra of Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, and Super Bright 780 polymer dyes. The black bar indicates the excitation wavelength of the violet laser (405 nm). The less-intense, brown curve under the blue emission shows the contribution of the (donor) Super Bright 436 dye to the emission curves of the four tandem Super Bright polymer dyes.

Figure 2. 10-color T cell subset panel. Human peripheral blood cells were diluted with Invitrogen eBioscience Super Bright Staining Buffer (Cat. No. SB-4400-42) and surface stained with the indicated reagents. Samples were then fixed and permeabilized according to the Invitrogen eBioscience Foxp3/Transcription Factor Staining Buffer Set protocol and stained with the indicated intracellular reagents. The fluorescent antibody panel included: anti-CD3 (clone OKT3) Super Bright 645, anti-CD4 (clone RPA-T4) eFluor 506, anti-CD8a (clone RPA-T8) APC-eFluor 780, anti-CD20 (clone 2H7) PE-Cyanine5.5, anti-CD25 (clone BC96) Super Bright 600, anti-CD27 (clone O323) Super Bright 436, anti-CD183 (CXCR3) (clone CEW33D) PE-eFluor 610, anti-CD185 (CXCR5) (clone MU5UBEE) PE-Cyanine7, anti-Foxp3 (clone PCH101) FITC, and anti-TIGIT (clone MBSA43) APC. Analysis was performed to discriminate various T cell subpopulations.

Super Bright polymer dyes

The Super Bright dyes are polymer-based fluorophores that are excited by the violet laser (405 nm) and named according to their emission wavelength (Figure 1). In addition to their very bright fluorescence emission, the Super Bright antibody conjugates are fully compatible with other fluorophores commonly used in flow cytometry—including R-phycoerythrin (PE), allophycocyanin (APC), and Invitrogen Alexa Fluor and eFluor dyes—as well as with Invitrogen eBioscience buffers and fixatives and Invitrogen UltraComp eBeads microspheres. These features, combined with our extensive portfolio of cell analysis reagents, provide many choices when designing multicolor flow cytometry workflows, while also allowing you to expand the utility of your violet laser.

The Super Bright 436 dye has an excitation maximum of 414 nm and an emission peak of 436 nm (Figure 1). We recommend using a 450/50 nm bandpass filter or equivalent, similar to that used for detection of eFluor 450 conjugates. Super Bright 436 antibody conjugates are brighter than eFluor 450 conjugates and can serve as an alternative for Brilliant Violet™ 421 conjugates, providing similar resolution of positive and negative populations (Figure 3). Stability studies indicate that the Super Bright 436 dye exhibits minimal loss of fluorescence when cells are exposed to formaldehyde fixative for up to 3 days or when cells are exposed overnight to ambient light.

The Super Bright 600 dye is a tandem dye comprising the Super Bright 436 dye and an acceptor dye that emits fluorescence at 600 nm (Figure 1). It can be detected using a 610/20 nm bandpass filter or equivalent. Super Bright 600 conjugates are comparable in brightness to Brilliant Violet™ 605 conjugates (Figure 4). The Super Bright 600 dye is stable for up to 3 days when stored in a formaldehyde fixative solution.

Super Bright 645 is a tandem dye consisting of Super Bright 436 and an acceptor dye that has an emission peak of 645 nm (Figure 1). It can be detected using a 660/20 nm bandpass filter or equivalent. This tandem polymer dye is comparable in brightness to Brilliant Violet™ 650 dye (Figure 5) and demonstrates less spillover into other violet channels. Super Bright 645 is stable for up to 3 days when stored in a formaldehyde fixative solution.

Super Bright 702 is a dye consisting of Super Bright 436 and an acceptor dye that has an emission peak of 702 nm (Figure 1). It can be detected using a 710/50 nm bandpass filter or equivalent, similar to Brilliant Violet™ 711 dye. Super Bright 702 antibody conjugates are similar in brightness to those of Brilliant Violet 711 dye (Figure 6) and feature less spillover into the Brilliant Violet™ 786 channel. Fixation compatibility is similar to the other Super Bright conjugates.

Super Bright 780 dye is an alternative to Brilliant Violet™ 786 dye (Figure 7). A tandem of Super Bright 436 and an acceptor dye with a 780 nm peak emission (Figure 1), Super Bright 780 dye can be detected using a 780/60 bandpass filter or equivalent in flow cytometry and has less spillover into other violet laser channels.

 Fluorescence intensity comparison with Super Bright 436 dye

Figure 3. Fluorescence intensity comparison with Super Bright 436 dye. (A) Human peripheral blood cells stained with CD19 antibody (clone HIB19) conjugated to either Super Bright 436 dye (purple histogram), eFluor 450 dye (blue histogram), or Brilliant Violet 421 dye (orange histogram) using the manufacturer’s recommended volume per test (cells were gated on lymphocytes). (B) Human peripheral blood cells stained with CD27 antibody (clone O323) conjugated to either Super Bright 436 dye (purple histogram) or Brilliant Violet 421 dye (blue histogram).

Staining performance and post-fixation stability of Super Bright 600 dye

Figure 4. Staining performance and post-fixation stability of Super Bright 600 dye. (A) Direct comparison of mouse splenocytes stained with CD8a antibody (clone 53-6.7) conjugated to either Super Bright 600 dye (red histogram) or Brilliant Violet 605 dye (gray histogram), at the same antibody concentration. (B) Mouse splenocytes stained with CD45R antibody (clone RA3-6B2) conjugated to Super Bright 600 dye and left unfixed (red histogram), or fixed in Invitrogen eBioscience IC Fixation Buffer for 30 min (blue histogram), 24 hr (orange histogram), or 3 days (green histogram).

Fluorescence intensity comparison with Super Bright 645 dye

Figure 5. Fluorescence intensity comparison with Super Bright 645 dye. (A) Mouse splenocytes stained with CD8a antibody (clone 53-6.7) conjugated to Super Bright 645 dye (red histogram) or Brilliant Violet 650 dye (gray histogram), at the same concentration of antibody. (B) Human peripheral blood cells stained with CD8a antibody (clone RPA-T8) conjugated to Super Bright 645 dye (red histogram) or Brilliant Violet 650 dye (gray histogram), using the same concentration of antibody.

Fluorescence intensity comparison with Super Bright 702 dye

Figure 6. Fluorescence intensity comparison with Super Bright 702 dye. (A) Mouse splenocytes stained with CD4 antibody (clone GK1.5) conjugated to Super Bright 702 dye (red histogram) or Brilliant Violet 711 dye (gray histogram), at the same concentration of antibody. (B) Human peripheral blood cells stained with CD19 antibody (clone HIB19) conjugated to Super Bright 702 dye (red histogram) or Brilliant Violet 711 dye (gray histogram), using the same concentration of antibody.

Fluorescence intensity comparison of Super Bright 780 conjugates and Brilliant Violet™ 786 conjugates

Figure 7. Fluorescence intensity comparison of Super Bright 780 conjugates and Brilliant Violet™ 786 conjugates. (A) Mouse splenocytes stained with anti-CD4 conjugated to Super Bright 780 (red, Cat. No. 78-0042-80) or Brilliant Violet™ 786 conjugate (gray), at the same concentration of antibody. (B) Human peripheral blood cells stained with anti-CD8a conjugated to Super Bright 780 dye (red, Cat. No. 78-0088-41) or Brilliant Violet™ 786 conjugate (gray), using the same concentration of antibody.

Super Bright staining buffer

Similar to traditional fluorescent conjugates, Super Bright antibody conjugates can be employed in most flow cytometry applications without adjusting protocols. However, if two or more Super Bright conjugates are combined in the same panel, we recommend using Invitrogen eBioscience Super Bright Staining Buffer to minimize any nonspecific interactions that may occur between these polymer-based dyes (Figure 8). No special buffer is required when using a single Super Bright conjugate within a panel that does not contain other polymer dye conjugates. When Super Bright conjugates are used in combination with other polymer dye conjugates, such as Brilliant Violet dyes, the Super Bright Staining Buffer can also be used to help reduce dye–dye interactions. Super Bright Staining Buffer is formulated for use at 5 μL/test, making it convenient when preparing cocktails.

Super Bright Staining Buffer minimizes nonspecific interactions

Figure 8. Super Bright Staining Buffer minimizes nonspecific interactions. Human peripheral blood cells were stained with CD8a antibody (clone RPA-T8) conjugated to Super Bright 600 and CD4 antibody (clone SK3) conjugated to Super Bright 436 (A, B) or Brilliant Violet 421 dye (C, D). Cells were stained in the presence of Invitrogen eBioscience Flow Cytometry Staining Buffer only (A, C) or Super Bright Staining Buffer was added to cells prior to addition of antibodies (B, D).

Expand the utility of the violet laser

The Invitrogen Attune NxT Flow Cytometer, introduced with up to 4 lasers and 16 parameters of detection, will soon be available with 6 fluorescence detectors for the violet laser, allowing measurement of up to 8 fluorescent parameters with a 2-laser instrument, 11 fluorescent parameters with a 3-laser instrument, or 14 fluorescent parameters with a 4-laser instrument (Table 1). The 6-channel violet laser configuration, available on the 2-, 3-, and 4-laser instruments, will accommodate the Super Bright polymer dye antibody conjugates along with other commercially available violet-excitable fluorophores (Table 2).

Table 1. Attune NxT Flow Cytometer configuration using 6 fluorescence detectors for the violet laser.

Target Fluorescence detectors available
2-laser configuration 3-laser configuration 4-laser configuration
Violet, 405 nm 6 6
6
Blue, 488 nm 2 2 2
Yellow, 561 nm NA NA 3
Red, 637 nm NA 3 3
Total fluorescence
detectors available per
configuration
8 11 14
Total parameters per
configuration (includes
FSC and SSC)
10 13 16

Table 2. Fluorophore guidelines for the 6 fluorescence detectors of the violet laser in the Attune NxT Flow Cytometer.

Detector Bandpass (nm) Fluorophores
VL1 450/40 Super Bright 436, Brilliant Violet 421, eFluor 450, Pacific Blue,
BD Horizon V450, VioBlue
VL2 525/50 eFluor 506, Brilliant Violet 510, Pacific Green, BD Horizon V500, VioGreen
VL3 610/20 Super Bright 600, Brilliant Violet 605, Pacific Orange
VL4 660/20 Super Bright 645, Brilliant Violet 650
VL5 710/50 Super Bright 702, Brilliant Violet 711
VL6 780/60 Super Bright 780, Brilliant Violet 786

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