StarBright Yellow 561: A reliable alternative to PE-tandem dyes

This article is based on a poster originally authored by S. Sanderson, L. Huang, M. Blundell, and T. Long, which was presented at ELRIG Drug Discovery 2024 in affiliation with Bio-Rad Laboratories, Inc.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

A well-designed panel with optimal fluorophore combinations is critical when generating multiplexing data. Best practice for panel design ensures that fluorophores that utilize all the available laser lines are chosen, thereby minimizing spillover and spread. However, this is difficult to achieve for the 561 nm (yellow) laser as the choice of fluorophores is limited.

Phycoerythrin (PE) and PE tandems are commonly used, but they are not the best choice for all assays.

Antibodies conjugated to StarBright^TM Yellow Dyes from Bio-Rad offer an alternative option. They are bright, with high stain index values (Figure 1), and reduced 488 nm (blue) cross-laser excitation than PE and PE-tandems (Figure 2).

This study shows multiplexing panels that incorporate antibodies conjugated to StarBright Yellow Dyes or PE/PE tandems using the yellow and blue lasers simultaneously. The StarBright Yellow Dye panel has less compensation and spread, resulting in improved population resolution and easier identification of cell populations (Figure 3).

They have less nonspecific binding to monocytes than tandem dyes containing cyanine (Figure 4). Unlike PE and PE-tandem dyes, StarBright Yellow Dyes can be fixed in both alcohol- and paraformaldehyde-based fixatives with no change in spectral profile, making them suitable for a wider range of assays (Figure 5).

These features make StarBright Yellow Dyes a superior choice over common alternatives, resulting in improved reproducibility and panels with reduced nonspecific binding and low spread, generating data with highly resolved cell populations.

Bright dyes

Fig. 1. Stain index values of mouse anti-human CD4 conjugated to StarBright Yellow Dyes (red) and other fluorescent dyes (blue). Red blood cell lysed human peripheral blood was stained with fluorescently labeled mouse anti-human CD4 and acquired on a ZE5 Cell Analyzer (Bio-Rad). Cells were gated on live, single-cell lymphocytes and the stain index calculated. Data shown as 3 donors +/- SD. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories

Reduced cross-laser excitation and improved cell resolution

StarBright Dyes are designed to have narrower excitation and emission profiles compared to alternative fluorophores. SBY575 has less cross-laser excitation than PE (Figure 2). This results in a reduction of spread and improved cell resolution (Figure 3).

Fig 2. Emission profiles of StarBright Yellow Dyes (red) and fluorophores of similar max excitation wavelengths (blue). Red blood cell lysed human peripheral blood was stained with mouse anti-human CD4 and data acquired on a 5- L Cytek Aurora. Graphs show normalized spectral profiles. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories

Fig. 3. Panels including StarBright Yellow Dyes or alternative PE and PE-tandem dyes with similar excitation and emission max. Red blood cell lysed human peripheral blood was stained with the antibody panels (Table 1) and a L/D dye (DAPI, Bio-Rad #1351303) and acquired on a ZE5 Cell Analyzer. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). Cells were gated on live, single cells. Populations with high spread in the PE/PE-tandem panels are highlighted in blue. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories

Table 1. Bio-Rad antibodies used for StarBright Dye and PE/PE-tandem panels. Source: Bio-Rad Laboratories

Reduced monocyte binding

Fig. 4. Binding of mouse anti-human CD3 conjugated to StarBright Yellow Dyes and other fluorescent dyes to human peripheral blood. Red blood cell lysed human peripheral blood was stained with fluorescently labeled mouse anti-human CD3 and acquired on the ZE5 Cell Analyzer. Cells were gated on live, single cells. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories

Fixable with PFA and alcohol

Fig. 5. Effect of fixatives on StarBright Yellow Dye conjugated antibodies compared to PE and PE-tandems. Human PBMCs were stained with mouse anti-human CD4 and acquired on a ZE5 Cell Analyzer before fixation, after fixation in a PFA-based fixation buffer (#00- 8222-49, Thermo Fisher Scientific) for 15 min, and after fixation in a PFA-based fixation buffer followed by incubation in 100 % MeOH for 30 min. Cells were gated on live, single-cell lymphocytes. Histogram overlays and the signal from CD4-positive cells in each filter are shown. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories

Conclusions

• StarBright Yellow Dyes are five new types of bright dyes that have improved spectral characteristics over PE and PE tandems due to reduced excitation by the 488 nm laser.
• When used in a simple immunophenotyping panel, there is reduced spreading compared to a panel containing PE and PE tandems with minimal reduction of signal due to a lower stain index.
• StarBright Yellow Dyes do not exhibit any nonspecific binding on monocyte populations often seen with cyanine dye containing tandems.
• StarBright Yellow Dyes can be fixed in PFA and alcohol with minimal loss of signal and no spectral changes, unlike PE and PE tandem dyes, which lose signal and exhibit severe spectral changes.

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