Unlock more colors in flow cytometry with new StarBright dyes

This article and associated images are based on a poster originally authored by Sharon Sanderson, Michael Blundell, and Tracey Long and presented at ELRIG Drug Discovery 2025 in affiliation with Bio-Rad Laboratories.

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.

The advancement of full-spectrum cytometry has resulted in the creation of more intricate multiplexing panels. However, the shortage of new dyes tailored for spectral instruments poses challenges in optimizing and broadening their applications. Bio-Rad is enhancing its line of 32 StarBright Dyes for flow cytometry by introducing four additional dyes: StarBright Violet 535, StarBright UltraViolet 540, StarBright UltraViolet 700, and StarBright UltraViolet 770.

This article presents findings on the Cytek Aurora (Cytek Biosciences) and ID7000 (Sony Biotechnology), showcasing that these bright, innovative dyes possess unique spectral profiles with narrow excitation and emission wavelengths, resulting in minimal spreading and high-quality multiplexing data.

Like all StarBright Dyes, these new additions do not require any special staining buffer, can be fixed without losing signal strength, and can be stored long term as a premixed panel, offering flexibility for both panel design and future expansion.

New StarBright dyes

Fig. 1. Spectral profiles of StarBright UltraViolet and Violet Dyes. Emission profiles of Mouse anti-Human CD4 StarBright UltraViolet and Violet conjugated antibodies generated on a 5L Cytek Aurora (Cytek Biosciences) and a 5L ID7000 (Sony Biotechnology). New spectral dyes are highlighted in red. Image Credit: Image courtesy of Sharon Sanderson et al., in partnership with ELRIG (UK) Ltd.

Materials and methods

Table 1. Bio-Rad antibodies used in the multiplex panel. Source: ELRIG (UK) Ltd. 

Staining conditions

Red blood cell lysed human peripheral blood was blocked with 10 % human serum and stained with VivaFix353/442 (#1351111, Bio-Rad Laboratories), if required.

After washing and resuspending, cells were stained with a single antibody or antibody panel in PBS + 1 % BSA or Brilliant Buffer (#00-4409-42, Thermo Fisher Scientific).

Cells were stained in a 96-well plate for one hour at room temperature (RT), washed three times, and fixed in Fixation Buffer (#BUF071, Bio-Rad) before resuspending in phosphate-buffered saline (PBS).

Immunophenotyping staining panel

All antibodies were titrated to determine the optimal staining concentration prior to use. Antibodies used in the panel are shown in Table 1. 

Data collection and analysis

Cells were acquired on a 5-laser Cytek Aurora or a 5-laser Sony ID7000 cytometer. Data was analyzed using FCS Express 7 (De Novo Software by Dotmatics).

Performance of new spectral dyes

Fig. 2. Similarity scores and Stain Index of StarBright UltraViolet and Violet Dyes. Human PBMCs stained with Mouse anti Human CD4 conjugated antibodies. Data generated on A. Cytek Aurora B. Sony ID7000. Image Credit: Image courtesy of Sharon Sanderson et al., in partnership with ELRIG (UK) Ltd.

Human immunophenotyping panel acquired on a 5L Cytek Aurora

Fig. 3. Human immunophenotyping panel acquired on a 5L Cytek Aurora. Human peripheral blood was stained with a 33-color antibody panel (Table 1). Image Credit: Image courtesy of Sharon Sanderson et al., in partnership with ELRIG (UK) Ltd.

Human immunophenotyping panel acquired on a 5L ID7000

Fig. 4. Human immunophenotyping panel acquired on a 5L Sony ID7000. Human peripheral blood was stained with a 32-color antibody panel (Table 1). CD45RASBY720 was accidentally omitted, therefore naïve and memory T cell populations could not be identified in this panel. Bio-Rad gratefully acknowledges the assistance of Jamie Evans, Flow Cytometry Core Facility Manager, Division of Medicine, Rayne Building UCL for providing access and assistance with their ID7000. Image Credit: Image courtesy of Sharon Sanderson et al., in partnership with ELRIG (UK) Ltd.

Conclusion

Four new StarBright Dyes have been developed to address gaps in the spectral range where no comparable dye with similar emission is available.

These dyes are bright and work well with the existing collection of 32 StarBright Dyes, as well as other frequently used fluorescent dyes in flow cytometry on both the Cytek Aurora and Sony ID7000.

When used in a 33-color panel, multiple lineages and subsets could be identified in human peripheral blood.

Retaining all the advantages of the StarBright Dyes, these new dyes are perfect for enhancing current spectral panels and provide greater flexibility when assembling panels of any dimension.

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Last Updated: Nov 26, 2025