Antibody conjugates for the violet laser

Invitrogen eBioscience Super Bright dyes are a series of fluorophores that are based on a fluorescent polymer and its tandems, named for their emission wavelength and excited by the violet laser (405 nm). These dyes are optimized for use in flow cytometry and may allow for better discrimination of dim populations due to their brightness. Super Bright antibody conjugates increase your options for multicolor panel design and allow you to expand the utility of your violet laser.

On this page:

Super Bright 436 dye

Invitrogen eBioscience Super Bright 436 dye has an excitation maximum of 414 nm and an emission peak of 436 nm. A 450/50 bandpass filter or equivalent is recommended, similar to Invitrogen eFluor 450 dye or Invitrogen Pacific Blue dye. Super Bright 436 dye–conjugated antibodies are significantly brighter than those conjugated to eFluor 450 dye, and are an alternative to Brilliant Violet 421 conjugates with similar resolution of positive and negative populations. Stability studies indicate that Super Bright 436 dye exhibits a minimal loss of fluorescence when stained cells are exposed to formaldehyde fixative for up to three days or left uncovered overnight in ambient light.

Figure 1. Super Bright 436 dye performance comparison. Mouse bone marrow cells were stained with Anti-Ly-6A/E (clone D7) APC and either Anti-CD117 (clone 2B8) conjugated to (left panel) Super Bright 436 dye, (middle panel) eFluor 450 dye or (right panel) Brilliant Violet 421 dye.


Super Bright 600 dye

Invitrogen eBioscience Super Bright 600 dye is a tandem dye comprising Super Bright 436 dye and an acceptor dye that emits at 600 nm. It can be detected using a 610/20 bandpass filter. Antibodies conjugated to this tandem polymer dye are comparable in brightness to Brilliant Violet 605 dye conjugates. Super Bright 600 dye–conjugated antibodies are stable for up to three days when stored in a formaldehyde fixative solution.  

Multiplexing with Super Bright 600 dye

Figure 2. Multiplex flow cytometry experiment. Normal human peripheral blood cells were diluted in Super Bright Staining Buffer, then surface stained with the indicated reagents. Viable cells (determined using a fixable viability stain) were used for analysis to discriminate various T cell subsets.


Super Bright 645 dye

Invitrogen eBioscience Super Bright 645 dye is a tandem dye consisting of Super Bright 436 and an acceptor dye that has an emission peak of 645 nm. It can be detected using a 660/20 bandpass filter or equivalent. Antibody conjugates of this tandem polymer dye are comparable, and sometimes superior in brightness to Brilliant Violet 650 antibody conjugates (Figure 3) with less spill over into other violet channels.

Invitrogen eBioscience Super Bright 645 dye

Figure 3. Fluorescence intensity comparison of Super Bright 645 conjugates and Brilliant Violet 650 conjugates. (A) Mouse splenocytes stained with anti-CD8a conjugated to Super Bright 645 (red, Cat. No. 64-0081-82) or Brilliant Violet 650 conjugate (gray), at the same concentration of antibody. (B) Human peripheral blood cells stained with anti-CD8a conjugated to Super Bright 645 (red, Cat. No. 64-0088-42) or Brilliant Violet 650 conjugate (gray), using the same concentration of antibody.


Super Bright 702 dye

Super Bright 702 tandem dye, consists of Super Bright 436 and an acceptor dye that has an emission peak of 702 nm. Conjugates can be detected using a 710/50 bandpass filter or equivalent, and Super Bright 702 dye–conjugated antibodies are similar in brightness to Brilliant Violet 711 conjugates (Figure 4) with reduced compensation and less spillover into the Brilliant Violet 786 channel.

Figure 4. Fluorescence intensity comparison of Super Bright 702 conjugates to Brilliant Violet 711 conjugates. (A) Mouse splenocytes stained with anti-CD4 conjugated to Super Bright 702 (red) or Brilliant Violet 711 conjugate (gray), at the same concentration of antibody. (B) Human peripheral blood cells stained with anti-CD19 conjugated to Super Bright 702 (red) or Brilliant Violet 711 conjugate (gray), using the same concentration of antibody.


Super Bright Staining Buffer

As with other polymer dyes, nonspecific dye–dye interactions may form when multiple Super Bright dye–conjugated antibodies are used together in the same experiment. When they occur, these interactions can cause unexpected population shifts. Use of the Super Bright Staining Buffer (Cat. No. SB-4400-42) prior to addition of Super Bright conjugates alleviates this issue and returns populations to their expected position within the plot. When using Super Bright dyes in combination with other polymer dyes, such as Brilliant Violet dyes, the Super Bright Staining Buffer can be used to minimize dye–dye interaction. Super Bright Staining Buffer is formulated to be used at 5 µL/test, making it convenient for use when preparing cocktails.

Super Bright Staining Buffer minimizes non-specific interactions

Figure 5. Super Bright Staining Buffer mitigates non-specific polymer dye interactions. (A) Flow Cytometry Staining Buffer (top plot) or Super Bright Staining Buffer (bottom plot) was added to human peripheral blood cells prior to staining with Anti-CD8a (clone RPA-T8) Super Bright 600 dye and Anti-CD4 (clone SK3) Super Bright 436 dye. (B) Flow Cytometry Staining Buffer (top plot) or Super Bright Staining Buffer (bottom plot) was added to human peripheral blood cells prior to staining with Anti-CD8a (clone SK1) Brilliant Violet 605 dye and Anti-CD4 (clone SK3) Super Bright 436 dye. (C) Flow Cytometry Staining Buffer (top plot) or Super Bright Staining Buffer (bottom plot) was added to human peripheral blood cells prior to staining with Anti-CD8a (clone RPA-T8) Super Bright 600 dye and Anti-CD4 (clone SK3) Brilliant Violet 421 dye.

Ordering information

Dye Super Bright 436 Super Bright 600 Super Bright 645 Super Bright 702
Excitation laser (nm) Violet (405) Violet (405) Violet (405) Violet (405)
Em (nm) 436 600 645 702
Suggested bandpass filter 450/50 610/20 660/20 710/50
Application Flow cytometry Flow cytometry Flow cytometry Flow cytometry
Comparable to Brilliant Violet 421 Brilliant Violet 605 Brilliant Violet 650 Brilliant Violet 711
Primary antibodies Super Bright 436 conjugates Super Bright 600 conjugates Super Bright 645 conjugates Super Bright 702 conjugates
Control antibodies Super Bright 436 conjugates Super Bright 600 conjugates Super Bright 645 conjugates Super Bright 702 conjugates
Super Bright Staining Buffer (Cat. No. SB-4400-42) is recommended for use with Super Bright polymer dyes.

Resources

Custom Services
Contact our eBioscience custom services group for antibody conjugation, antibody cocktails, immunoassay development, bulk reagents, and testing services.

Support