Spectral Flow Cytometry Introduction

Spectral flow cytometry was first proposed in 1979 (Wade et al. 1979) and a first functional spectral flow cytometer presented at the International Society for Analytical Cytology in 2004 by J P Robinson (Robinson et al. 2004), with a patent issued to Purdue University in 2007 (Robinson 2019). Unlike conventional flow cytometry that partitions the emitted light from laser excited fluorophores onto specific detectors, spectral flow cytometry uses prisms to collect all the emitted light across an array of detectors. This allows capture of the entire spectral profile or signature from multiple lasers. The profiles of the fluorophores are then unmixed using complex mathematical models to separate and identify each fluorophore.

Spectral Analyzer

Spectral Analyzer

 

One advantage of spectral over conventional flow cytometry is the ability to use fluorophores with a similar peak emission, providing they have a distinct signature across the full spectrum. An example of this is APC and Alexa Fluor 647. These fluorophores cannot be used together in conventional flow cytometry, but the emission profile from the violet laser differs enough for them to be separated in spectral flow cytometry (Ferrer-Font et al. 2020). This technique allows the size of multicolor immunophenotyping panels to be extended to over 40 markers.

The same principles of good multicolor panel design are also required for spectral flow cytometry. Some knowledge of the biology of your experiment such as antigen density, the fluorophores, including brightness and optimization of your panel are still required. Ferrer-Font et al. (Ferrer-Font et al. 2020) have recently published in-depth information on panel design for spectral flow cytometry, Panel Design and Optimization for High‚ÄźDimensional Immunophenotyping Assays Using Spectral Flow Cytometry in Current Protocols in Cytometry.


Using StarBright Dyes in Spectral Flow Cytometry

New StarBright Dyes are proprietary, fluorescent nanoparticles developed specially for flow cytometry. They are bright with exacting excitation and emission characteristics. They can be used in all common staining buffers without the need for special buffers, have a high stability, and low lot-to-lot variation. We compared the performance of these dyes in conventional and spectral flow cytometry and found a very similar performance in both, with good separation of populations and percent identified as positive (Figure 1).

Fig. 1. Comparison of conventional and spectral flow cytometry


Fig. 1. Comparison of conventional and spectral flow cytometry. Red blood cell lyzed human peripheral blood was blocked with 10% human serum and then stained in 1% BSA in PBS with CD3SBB700 (MCA463SBB700), CD4SBV515 (MCA1267SBV515), CD8SBV610 (MCA1226SBV610), CD19SBV670, CD25SBV440 (MCA2127SBV440), and CD127A647 (HCA145A647) for 30 min. B cells, helper, cytotoxic, and regulatory T cells could be distinguished from each other. There were minimal differences between A, conventional flow cytometry and B, spectral flow cytometry in the populations seen, and the percentages obtained.


Spectral Profiles of StarBright Dyes

Fig. 2. Spectral profiles of StarBright Dyes.


Fig. 2. Spectral profiles of StarBright Dyes. Spectral profiles of antibodies conjugated to StarBright Dyes, generated on a four-laser (16UV-16V-16B-10R) Aurora Spectral Analyzer (Cytek). 


Benefits of StarBright Dyes in Spectral Flow Cytometry

One advantage of spectral flow cytometry is the ability to use fluorescent dyes with similar emission peaks together in a multicolor panel. This has led to the creation of 40+-color panels, whereas conventional flow cytometry is currently limited to around 30 colors. Many StarBright Dyes have a unique spectra, therefore novel combinations can be made with dyes that have similar maximal emission peaks. Examples of compatible dyes are StarBright Blue 700 (SBB700) Dye and PerCP-Cy5.5, StarBright Violet (SBV) 400 Dye with Brilliant Violet (BV) 421 and Pacific Blue, SBV475 with SBV515 and BV510, SBV610 and BV605, and SBV710 with BV711 (Figure 3).

Fig. 3. Examples of novel dye combinations to stain human peripheral blood.


Fig. 3. Examples of novel dye combinations to stain human peripheral blood. A, CD20PerCP-Cy5.5 in combination with CD45ROSBB700 (MCA461SBB700), B, CD4BV421, CD8Pacific Blue (MCA1226PB), and CD3SBV440 (MCA463SBV440), C, IgDBV510, CD8SBV475 (MCA1226SBV475), and CD2SBV515 (MCA1194SBV515), D, CD4BV605 and CD8SBV610 (MCA1226SBV610), and E, TCR gdBV711 and CD14SBV710 (MCA1568SBV710). 

Although many StarBright Dyes can be used with fluorescent dyes with similar maximal emission, enabling bigger panels to be created, many of the similarity scores are high, leading to excessive spreading. Therefore, best practice in panel design should always be followed. Separation of these dyes onto mutually exclusive markers can help negate the effect of the spreading. The tables below show the similarity scores of StarBright Dyes compared to Brilliant Ultraviolet (BUV), Brilliant Violet (BV), and Brilliant Blue (BB) dyes.

Table 1. StarBright Dye similarity scores.

StarBright UltraViolet Dye

Competitor Dye

Similarity Score

SBUV400

BUV395

0.99

SBUV445

 

 

SBUV510

BUV496

0.91

SBUV575

BUV563

0.95

SBUV605

BUV615

0.88

SBUV665

BUV661

0.76

SBUV740 (coming soon)

BUV737

Unknown

SBUV795

BUV805

0.92

StarBright Violet Dye

Competitor Dye

Similarity Score

SBV440

BV421

0.84

SBV475

BV480

Unknown

SBV515

BV510

0.9

SBV570

BV570

Unknown

SBV610

BV605

0.94

SBV670

BV650

0.96

SBV710

BV711

0.96

SBV790

BV785

0.93

StarBright Blue Dye

Competitor Dye

Similarity Score

SBB700

BB700/

PerCP-Cy5.5

0.95

To help you build panels with StarBright Dyes, download the poster presented at CYTO2021, describing how we built a 27-color panel containing 13 StarBright Dyes and/or listen to an on-demand webinar on how to expand your spectral flow cytometry knowledge and panel size.

Click on the links below to find out more on each StarBright Dye.

Available Now


New StarBright Dyes Coming Soon


References


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