Enhancing flow cytometry data with stable StarBright dyes

This article is based on a poster originally authored by S. Sanderson, R. Cuthbert, M. Blundell, and T. Long, which was presented at ELRIG Drug Discovery 2024 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.

Fluorophore instability is a common issue that can affect data quality. Tandem dyes are well known to have poor photostability, causing them to break down into their constituent donor and acceptor molecules, altering their spectral profile.

Fixation can alter or completely degrade fluorophore profiles, resulting in a loss of signal. For example, alcohol-based fixatives can affect phycoerythrin (PE) and PE tandems in this way.

Multiplexing with these unstable fluorophores can impact conventional and spectral cytometry data quality. The performance of a well-designed panel giving excellent data and cell resolution may decline if any of the fluorophore’s spectral profiles are subsequently altered.

The panel may no longer be compensated or unmixed correctly if new controls using the degraded fluorophores were not acquired and the original algorithm was applied. Increased spreading can also occur, which results in poorer resolution of cell populations.

Not all fluorophores are compatible with every assay. Intranuclear and phosphoenol protocols contain an alcohol fixation step. Therefore, any fluorophores used in these assays must be able to withstand this method of fixation, reducing the fluorophore choice available.

The data presented here show that StarBrightTM Dyes (Figure 1) are highly stable, maintaining consistent spectral profiles and brightness. They show lot-to-lot and within-lot stability (Figure 2), with minimal variation in all standard staining buffers tested (Figure 3) and after fixation in both PFA and alcohol-based fixatives (Figure 4).

They also have the same or improved photostability as alternative fluorophores (Table 1 and Figure 5). This improved spectral stability means consistent data is generated, with no increased spillover and spread introduced from dye breakdown.

StarBright UltraViolet, Violet, Blue, Yellow, and Red Dye range

StarBright Dyes are a range of 32 superior dyes excited by the most common laser lines: 355 nm, 405 nm, 488 nm, 561 nm, or 640 nm.

Fig. 1. Emission spectra for StarBright Dyes. StarBright Dyes excitable by A, the 355 nm laser; B, the 405 nm laser; C, the 488 nm laser; D, the 561 nm laser; E, the 640 nm laser. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories.

Lot stability

Fig. 2. Consistent staining using StarBright Dye conjugated antibodies within and between lots. Red blood cell lysed human peripheral blood was stained with mouse anti-human CD3 (MCA463SBB700) or mouse anti-human CD4 (MCA1267SBV515) conjugated to StarBright Dyes and acquired on a 5 laser ZE5 Cell Analyzer (Bio-Rad). Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories.

Suitable for all buffers

StarBright Dyes are compatible with common staining buffers, as shown by a consistent brightness and stable spectral profile. An example of a StarBright Dye excited by each laser line is shown below.

Fig. 3. Brightness and spectral stability of StarBright Dye conjugated CD4 antibodies after staining in common buffers. Red cell lysed human peripheral blood was stained with mouse anti-human CD4 (MCA1267SBUV400, MCA1267SBV515, MCA1267SBB580, MCA1267SBY605 or MCA1267SBR670) for 1 hr in PBS + 1% BSA, Bio-Rad Stain buffer (#BUF073) or Brilliant stain buffer (BD Biosciences, #563794). Cells were acquired on a 5-laser ZE5 Cell Analyzer. Histogram overlays and the spectral profile from CD4 positive cells in each ZE5 Cell Analyzer filter are shown as a ratio of the signal in their target filter. Different donors were used. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories.

Fixable in PFA or alcohol

StarBright Dyes remain stable after fixation. The brightness after staining with a StarBright Dye-conjugated CD4 antibody is shown before and after fixation in common fixatives. An example of a StarBright Dye excited by each laser line is shown below.

Fig. 4. Stability of StarBright Dye conjugated antibodies after fixation. Red cell lysed human peripheral blood was stained with mouse anti-human CD4 (MCA1267SBUV510, MCA1267SBV670, MCA1267SBB675, MCA1267SBY605 or MCA1267SBR775) and acquired on a 5-laser ZE5 Cell Analyzer before and after fixation in Bio-Rad Fixation buffer (BUF071), 2 % PFA or 70 % EtOH. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories.

Photostability – Stain index

StarBright Dyes and other fluorophores conjugated to mouse anti-human CD4 were left out in the light for up to 14 days. As would be expected, many fluorophores lost brightness by day 14. StarBright Dyes show the same or improved stain index stability compared to comparison fluorophores.

Table 1 A-E. Photostability of the stain index of StarBright Dyes and other fluorophores conjugated to mouse anti-human CD4. Red blood cell lysed human peripheral blood was stained with antibodies left out 1, 4 or 14 days in light at room temperature and a control antibody stored at 4 °C in the dark. The stain index was calculated for each antibody after staining and expressed as a percentage of the control antibody. NT = not tested. Source: Bio-Rad Laboratories

A. 355 nm excitable fluorophores

B. 405 nm excitable fluorophores

C. 488 nm excitable fluorophores

D. 561 nm excitable fluorophores

E. 640 nm excitable fluorophores

Key

Photostability – Spectral profile

StarBright Dyes maintain a stable spectral profile when exposed to light, even with a reduced stain index. Spectral profiles at four and 14 days for StarBright Dyes and comparable dyes with similar maximum excitation and emission are shown alongside the control.

The profile for antibodies that lost all positive signals could not be determined. An example from each laser line is shown below.

Fig. 5. Spectral photostability of StarBright Dyes and other fluorophores conjugated to mouse anti-human CD4. Red blood cell lysed human peripheral blood was stained with antibodies left out 1, 4 or 14 days in light at room temperature or a control antibody stored at 4 °C in the dark. Cells were acquired on a 5-laser ZE5 Cell Analyzer. The stain index was calculated and expressed as a percentage of the control antibody. A spectral profile was generated by plotting the signal in each filter as a ratio of the signal in their target filter. Image Credit: S. Sanderson et al., in partnership with Bio-Rad Laboratories.

Conclusions

• StarBright Dyes are compatible with common buffers and fixatives, giving stable, reproducible results.
• They have improved photostability over many common fluorophores and maintain a stable spectral profile even when the signal is reduced by photobleaching. This allows consistent data to be generated, with minimal experimental changes observed/introduced from dye breakdown.
• StarBright Dyes are suitable for all protocols and ideal for multiplexing assays in both conventional and spectral flow cytometry.

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