Super Bright Fluorescent Polymer Dyes for the Violet Laser

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 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.

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.

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.

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