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 JP 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. 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 an in-depth information on panel design for spectral flow cytometry, Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry in Current Protocols in 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 flow and spectral flow cytometry and found very similar performance with good separation of populations and percent positive (Figure 1).
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.
As previously mentioned, one of the advantages of spectral flow cytometry is the ability to use fluorophores with similar emission peaks together in a multicolor panel. This advantage 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 and StarBright Violet 610 was thought to be sufficiently different to BV605 (Figure 2) to be used together in a panel. As can be seen in Figure 3, when combined in a small panel on CD4 and CD8, two distinct populations could be easily resolved.
Fig 2. Full spectra of SBV610 and BV605. Spectra obtained on the Cytek Aurora using SpectroFlo software.
Fig. 3. Spectral unmixing of SBV610 and BV605. Red blood cell lyzed human peripheral blood was blocked with 10% human serum and then stained in 1% BSA in PBS with CD3SBB700 (MCA463SBB700), CD4SBV610 (MCA1267SBV610), CD8BV605, and CD19A488 (MCA1940A488) for 30 min. B cells and T cells can be identified with helper and cytotoxic T cells distinguished from each other within the T cell population.
Find out more about StarBright Dyes and how they can add to your multicolor panel options.
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