Fluorescent Probe Technologies Suitable for Multicolor Spectral Flow Cytometry

news November 10 2021

Flow cytometry is a versatile tool that has applications in numerous biomedical research disciplines, including cancer biology, immunology, infectious disease monitoring, microbiology, and molecular biology. Instrumentation for performing flow cytometry has advanced considerably over the past several decades. Systems incorporating multiple lasers are commonplace, as are specialized instruments designed for particular applications, such as systems using 96-well sample loaders, systems combining microscopy with flow cytometry, and systems combining mass spectrometry with flow cytometry. Spectral flow cytometry measures the entire fluorescence emission spectra for each fluorophore deployed in a multicolor labeling experiment to produce unique spectral fingerprints. Subsequently, each spectrum is unmixed to provide a pure signal for each fluorophore. Such complete spectral analysis is beginning to supplant conventional photomultiplier tube (PMT)-based approaches as the preferred detection approach for high-dimensional multicolor flow cytometry. This technology shift provides an excellent opportunity to implement comprehensive, high-performance fluorophore families spanning the UV-visible-NIR light spectrum.

Multicolor Spectral Flow Cytometry Instrumentation

Flow cytometry is an analytical method that provides rapid, multi-parametric analysis of thousands to millions of individual cells in solution (McKinnon, 2018). Particular phenotypic characteristics of individual cells within a cell population are analyzed based upon their fluorescent and light scattering characteristics. Flow cytometry is commonly used in biomedical research and the analysis of clinical specimens. Flow cytometers employ lasers as light sources to generate both scattered and fluorescent signals that are registered by detectors, such as photodiodes or photomultiplier tubes. These analog signals are then converted into electronic signals, analyzed by a computer, and written to a standardized data file format (.fcs).

In order to expand upon the number of fluorophores employed in a flow cytometry experiment beyond ~28, a pretty high level of detail is required to distinguish among fluorophores whose spectral emission signatures are similar. The required level of detail demands high-quality signals, low noise, excitation-specific full emission profiles, and vigilant panel design and optimization. Spectral flow cytometry represents a novel flow cytometry technology platform offering significantly improved multiplexing capabilities relative to traditional flow cytometry (Robinson, 2019; Fox et al., 2020). While in some instances, conventional flow cytometers are capable of detecting panels containing as many as two dozen or so different fluorophores, spectral flow cytometers can distinguish as many as 40 different fluorophores using a single multi-parametric panel.

Spectral flow cytometry shares much of the same hardware that is associated with conventional flow cytometry. Both systems employ standard fluidic and laser technologies that enable cell-by-cell spectral analysis. For both spectral and conventional flow cytometry methods, the process begins by delivering a sample stream through a flow chamber where cells move in single file at a constant velocity, a process referred to as hydrodynamic focusing. This allows uniform and efficient excitation by a set of monochromatic lasers. Emitted photons are then detected and analyzed by a combination of optics and software to identify unique spectral emission information from fluorophore-labeled biomolecules.

Spectral flow cytometry differs noticeably from traditional flow cytometry when considering its optical configuration and the data analysis software (figure 1). With conventional flow cytometry, photons emitted by a particular fluorophore are directed through a series of dichroic mirrors and bandpass filters to partition the light into a narrow bandwidth for detection by a specific photomultiplier tube (PMT). However, spectral flow cytometry is different in that it employs dispersive optics, such as prisms or spectrographs to disperse photons based upon wavelength across an array of detectors. This approach broadens a fluorophore’s spectral profile by capturing the entire visible and near-IR spectrum of light, allowing for higher resolution spectral analysis relative to the aforementioned traditional optical configurations, which detect only a tiny portion of the emission spectra. As a result, fluorophores with similar emission spectral profiles that are challenging to differentiate by traditional flow cytometry can readily be distinguished using spectral flow cytometry. As many as 40 different fluorophores can be analyzed, including those with emission spectra in close proximity to one other.

Figure 1. Comparison of conventional and spectral flow cytometer systems. (Top) A conventional flow cytometer relies on a series of bandpass filters and dichroic mirrors to separate light emissions into individual detectors. (Bottom) A spectral flow cytometer uses a grating or prism element to separate light into a focusing lens prior to detection. As separating light keeps diverging in space, a collimating lens is often used to parallelize and direct light linearly before reaching a detector.

 

Despite its superior ability to distinguish between fluorophores, it is advisable to use fundamental flow cytometry principles in panel construction and avoid spectral spillover where possible when designing spectral flow cytometry experiments. Designing robust multicolor fluorescence panels entails vigilant consideration of fluorophore spectral properties, choosing those spectral profiles that are best separated by the instrument. Using traditional flow cytometry, any multi-parametric panel containing a dozen or more fluorophores will undoubtedly have some overlap of emission spectra, resulting in noise in channels from emissions of unintended fluorophores (i.e., spectral spillover). To account for this, single-color control samples must be included with each experiment to ascertain the level of spectral overlap encountered in each detector. Then, with traditional flow cytometry, a mathematical approach referred to as compensation is applied using the controls to subtract overlapping spectra and effectively isolate a fluorophore’s emission profile. This is achieved with a correction factor obtained using the ratio of the intended and unintended fluorophores emitting into a particular channel.

Mathematical models are required with spectral flow cytometry to correct for any spectral spillover among channels. This is necessary since the instrument must distinguish between multiple fluorescent profiles across the entire visible light spectrum rather than from a few distinct channels. The process of deconvoluting fluorophore emission spectra across an array of detectors is known as spectral unmixing. This form of compensation also requires experimental reference controls as well as noise-reducing mathematical algorithms, such as the least-squares method. This approach is especially valuable when interrogating cell culture samples, which are susceptible to a high degree of autofluorescence.

The Cytek Aurora benchtop flow cytometer (Fremont, CA) is a high-performance spectral flow cytometer that the AAT Bioquest team has extensive experience using in conjunction with our comprehensive suite of fluorophores and fluorescent proteins. The instrument leverages full-spectrum technology to enable the use of a wide range of novel fluorophore combinations without reconfiguring the system for each application. The instrument generates high-resolution data at the single-cell level, facilitating the resolution of even the most challenging cell populations, including cells exhibiting high autofluorescence or cells expressing low levels of target biomarkers, even in the context of complex multi-analyte detection. Their SpectroFlo® software provides an intuitive workflow that guides users from QC to data analysis with tools that simplify performing even the most challenging applications.

With up to five lasers, three scattering channels, and 64 fluorescence channels, the Aurora system is highly flexible, intuitive, and ultra-sensitive. With its intuitive optical design, compact footprint, and upgradeability from 3 to 5 laser configurations, the Aurora system suits every laboratory’s needs, from simple to high complexity applications.

The system’s state-of-the-art optics and low-noise electronics provide excellent sensitivity and resolution. Flat-top beam profiles, combined with a uniquely designed fluidics system, translate to outstanding instrument performance, even at high sample flow rates.

Table 1. Comparison between traditional and spectral flow cytometry.

Parameter Conventional Flow Cytometry Spectral Flow Cytometry
Spectral Resolution Acquires a narrow emission bandwidth from a single-laser excitation source Acquires the entire fluorophore emission profile for each laser excitation source
Optical Hardware Bandpass filters & dichroic mirrors
Capture a narrow portion of a fluorophore’s emission spectrum
Dispersive optics capture entire fluorophore’s emission spectrum
Multiplexing Capability Limited to perhaps 12 due to spectral overlap of fluorophores & limited number of detectors/filters installed in instrument Superior spectral resolution so that as many as 40 fluorophore probes can be employed in a single analysis
Fluorophore Separation Achieved by compensation (loss of some acquisition data) Achieved by spectral unmixing (preserves more acquisition data)
Detection Sensitivity Detection sensitivity is compromised by using compensation to address signal spill over Superior detection sensitivity is assured using state-of-the-art optics and low noise electronics

 

Optimized Multicolor Immunofluorescence Panels for Multicolor Spectral Flow Cytometry

A wide range of fluorescent reagents is employed in flow cytometry, including fluorescent dye-conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes, and different fluorescent proteins. By collecting substantially more information than conventional flow cytometry concerning each cell in a sample, full-spectrum multicolor flow cytometry has become the platform of choice for developing optimized multicolor immunofluorescence panels (OMIPs). Numerous OMIPs have been published in the literature over the years, obviating the need for extensive time to independently design such panels (Mahnke et al., 2010; Park et al., 2020). These published OMIPs also serve as valuable starting points for creating novel OMIPs and provide a mechanism for recognizing panel developers through citation of their work.

Table 2. Representative examples of optimized multicolor immunofluorescence panels (OMIPs).

Panel Plex Target Citation
OMIP-020 12 Phenotypic characterization of human γδT-cells Wistuba-Hamprecht et al., 2014
OMIP-037 16 Measuring inhibitory receptor signatures from multiple human immune cell subsets Belkina et al., 2017
OMIP-041 9 Phenotypic characterization of rat-derived microglial cells isolated from brain or spinal cord Toledano Furman et al., 2018
OMIP-050 28 Enumerating and characterizing cells expressing a wide array of immune checkpoint molecules Nettey et al., 2018
OMIP-069 40 Deep immunophenotyping of major cell subsets in human peripheral blood Park et al., 2020
OMIP-070 27 Deep immunophenotyping of major cell subsets in human peripheral blood Frutoso et al., 2020

 

As an impressive example of a biomedical research problem requiring the application of OMIPs, elucidating the intricate details of the human immune response requires the capacity to perform high-throughput, in-depth analysis at both the single-cell and population levels. OMIP-069 is a recently developed forty-color full spectrum flow cytometry panel designed for the deep immunophenotyping of major cell subsets in human peripheral blood, including subsets of T, B, NK, NKT, monocyte, and dendritic cells (Park et al., 2020). The cited panel shares similarities with OMIPs -015, -023, -024, -030, -033, -034, -042, -50, -058, -063, which were designed to identify the major leukocyte subsets in human blood, but also partially overlaps with OMIPs -013, -017, -021, -030, and -060 to facilitate characterization of T cells; OMIPs -004, -006, and -015 for Treg immunophenotyping; OMIP -044 for dendritic cells; OMIPs-003, -033, and -051 for B cells; and OMIPs -029, and -039 for NK cells. The panel includes a live-dead viability stain, a compendium of organic fluorophores, and various fluorescent proteins, such as allophycocyanin (APC), peridinin-chlorophyll-protein (PerCP), and various phycoerythrin (PE)-dye conjugates. The cited 40-color panel provides a potent approach for the in-depth characterization of lymphocytes, monocytes, and dendritic cells in human peripheral blood. The panel is suitable for interrogating nearly the entire cellular composition of the human peripheral immune system and should be particularly valuable for studies wherein sample availability is limited or unique biomarker signatures are being pursued.

Selecting Fluorophores for the Development of Multicolor Phenotypic Panels

In flow cytometry methods development, it is critically important to be familiar with the configuration of the instrument that a particular phenotypic panel is being designed for. The number and types of lasers and filters that the instrument is equipped with dictate which fluorophores can be deployed in the panel. Fluorophores are selected that have excitation maxima closely matching the lasers in the system, while for conventional flow cytometry filters should be designed to detect the targeted fluorophore’s emission wavelength maximum without registering light from other laser sources in the instrument. For multicolor spectral flow cytometry, an entire emission profile is detected, and fluorophores should be readily distinguishable on this basis. Typically, when a panel of fluorophores is required, distributing fluorophores as widely as possible across the excitation and emission spectra are recommended to minimize interference.

To minimize any signal spillover, which can adversely influence resolution and sensitivity, bright fluorophores, such as PE and APC, should be used with low abundance phenotypic targets, and organic fluorophores, such as mFluor™ dyes should be paired with more highly expressed targets. This improves the ability of the flow cytometer to discriminate between specific signal and background fluorescence arising from variables such as non-specific staining and cellular autofluorescence. In cases wherein an antibody is not commercially available bound to the desired fluorophore, ATT Bioquest offers custom antibody labeling kits and services.

The mFluor™ Dyes

As briefly summarized above, the foundation for developing multicolor phenotypic panels, such as the OMIPs, is a high-performance series of reactive organic fluorophores, fluorescent proteins, and tandem fluorophore-fluorescent protein conjugates that span the UV to near-infrared (NIR) region of the light spectrum. Designing and optimizing these panels can be a time-consuming and challenging activity. Several hurdles must be overcome, such as qualification (e.g., titration) of individual reagents, evaluating any potential interactions between reagents selected for a particular panel, choosing the best combination of fluorophore-antibody pairings, and overcoming the sensitivity losses inherent in multicolor fluorescence experiments. The design and optimization phase of creating even a modest 12 color panel may require 2–4 months of effort before the panel is mature enough for actual experimental applications. As a dedicated vendor in this specialized area of reagents, AAT Bioquest offers in-house developed conjugates, including custom-synthesized conjugates, to assist investigators in achieving the high-level multiplexing required for the development of multicolor immunofluorescence panels.

Developed exclusively by AAT Bioquest, mFluor™ dyes exhibit excellent aqueous solubility and large Stokes Shifts, and their hydrophilic nature minimizes the need for employing organic solvents (Table 3). Currently, we offer 26 unique mFluor™ SE dyes. mFluor™ dyes use the following naming convention: ‘mFluor™ + Excitation Laser + Emission Wavelength + Reactivity’. For example, mFluor™ UV460 SE (Ex/Em max = 364/461) is a UV light-excitable succinimidyl ester dye that emits around 460 nm.

mFluor™ dyes have been used extensively to label antibodies proteins and other biomolecules for multicolor flow cytometry applications. The absorbance maxima of mFluor™ dyes are designed to be optimally excited by one of the major laser lines commonly equipped in flow cytometers, such as the 350 nm, 405 nm, 488 nm, 532 nm, 561-568 nm, or 633-647 nm laser lines. In conjunction with phycobiliproteins PE, APC, and their tandems, mFluor™ dyes are excellent fluorophores for immunophenotyping (figure 2), FACS, and other flow cytometry-based applications. mFluor™ dyes are available in a wide selection of products, including reactive dyes and antibody labeling kits, as well as mFluor™ streptavidin conjugates for signal amplification and annexin V-mFluor™ conjugates for apoptosis detection.

Figure 2. Top) Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to generate four distinct emission profiles, then, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of whole blood cells stained with CD4-mFluor™ Violet 610 conjugate. The fluorescence signal was monitored using a Cytek® Aurora flow cytometer in the mFluor™ Violet 610 specific V10-A channel.

 

One crucial advantage of mFluor™ reactive dyes is that they can be covalently labeled to biomolecules without self-quenching, which results in intensely bright fluorescent conjugates. mFluor™ reactive dye formats include amine-reactive succinimidyl esters (SE) and thiol-reactive maleimides for labeling antibodies and proteins. Additionally, mFluor™ dyes are available as their acid form for labeling proteins, peptides, amine-modified oligonucleotides, and other amine-containing biomolecules using carbodiimide conjugation chemistry (EDAC).

To ensure stable, dependable antibody labeling for a variety of flow cytometry applications, antibody labeling kits can be an optimum choice for preparing the required antibody conjugates. ReadiLink™ Rapid mFluor™ Antibody Labeling Kits from AAT Bioquest provide a convenient method for labeling microscale volumes of antibodies with our superior mFluor™ dyes. The unique chemistry of ReadiLink™ kits enables researchers to effortlessly label and recover 100% of their antibodies without a purification step. Since ReadiLink™ mFluor™ conjugates are covalently labeled, they are stable for long-term storage and are ideal for demanding applications including immunophenotyping, multiplex flow cytometry, FACS, and other flow-based applications. Also available are ReadiLink™ Rapid and xtra Rapid Antibody Labeling kits for conjugation of mFluor™ dyes, Alexa Fluor® dyes, other fluorescent dyes, biotin, BSA, and KLH.

mFluor™ dyes provide simple, quick, and robust labeling of biomolecules, with high conjugation yields. The fluorophores offer superior photostability, solubility, and brightness generating strong fluorescence emissions over a broad pH range with little pH sensitivity and low spillover into most PMT detectors. As such, mFluor™ dyes offer excellent choices and flexibility for the design of multicolor phenotypic panels.

Table 3. mFluor™ active esters and kits for labeling antibodies, proteins and amine-modified biomolecules.

mFluor™ Dye Laser Ex max Em max ε¹ Φ² CF @260 CF @280 Succinimidyl Ester ReadiLink™ Kits
mFluor Blue 570 Blue 503 565 120,000 0.08 0.228 0.179 ABD-1160 ABD-1120
mFluor Blue 580 Blue 485 580 40,000 0.02 0.363 0.247 ABD-1178
mFluor Blue 590 Blue 500 589 81,000 0.15 0.671 0.406 ABD-1161
mFluor Blue 620 Blue 500 616 98,000 0.18 0.683 0.849 ABD-1163
mFluor Blue 630 Blue 470 634 49,000 0.015 0.197 0.275 ABD-1164
mFluor Blue 660 Blue 481 663 26,000 0.338 0.320 ABD-1180
mFluor Green 620 Blue/ Green 525 623 50,000 0.06 0.895 0.569 ABD-1165 ABD-1123
mFluor Green 630 Blue/ Green 537 657 51,000 ABD-1168
mFluor Red 700 Red 633 695 295,000 0.029 0.135 0.127 ABD-1190 ABD-1130
mFluor Red 780 Red 629 767 90,000 0.034 0.101 0.116 ABD-1191 ABD-1131
mFluor UV375 UV 351 387 30,000 0.94 0.099 0.138 ABD-1135
mFluor UV420 UV 353 421 80,000 ABD-1641
mFluor UV455 UV 357 461 20,000 0.42 0.651 0.406 ABD-1642
mFluor UV460 UV 358 456 15,000 0.86 0.35 0.134 ABD-1136
mFluor UV520 UV 370 524 80,000 0.03 0.495 0.518 ABD-1643
mFluor UV540 UV 373 560 90,000 0.35 0.634 0.463 ABD-1645
mFluor UV610 UV 371 609 90,000 0.25 0.949 0.904 ABD-1649
mFluor Violet 420 Violet 403 427 37,000 0.91 ABD-1105
mFluor Violet 450 Violet 406 445 35,000 0.81 0.338 0.078 ABD-1150 ABD-1100
mFluor Violet 500 Violet 410 501 25,000 0.81 0.769 0.365 ABD-1149
mFluor Violet 505 Violet 393 504 40,000 0.45 0.888 0.403 ABD-1154
mFluor Violet 510 Violet 412 505 25,000 0.86 0.464 0.366 ABD-1151 ABD-1110
mFluor Violet 540 Violet 402 535 18,000 0.21 1.326 0.543 ABD-1152 ABD-1114
mFluor Violet 545 Violet 393 543 20,000 0.15 1.080 0.496 ABD-1157
mFluor Violet 550 Violet 419 550 90,000 0.31 0.474 0.306 ABD-1153
mFluor Violet 590 Violet 424 591 90,000 0.22 0.632 0.329 ABD-1155
mFluor Violet 610 Violet 421 613 92,000 0.3 0.532 0.660 ABD-1156
mFluor Yellow 630 Green/ Yellow 570 632 110,000 0.01 0.283 0.413 ABD-1170 ABD-1126
  1. Extinction coefficient (M-1cm-1)
  2. Quantum yield measured in aqueous solution

 

Phycobiliproteins

Phycobiliproteins are photosynthetic light-harvesting proteins obtained from microalgae and cyanobacteria. This family of proteins contains covalently attached linear tetrapyrrole groups, referred to as phycobilins, which are involved in capturing light energy. In nature, energy absorbed by these phycobilins is efficiently transferred, by the process of fluorescence resonance energy transfer (FRET), to chlorophyll pigments for use in the photosynthetic process. Since phycobiliproteins possess extremely high fluorescence quantum yields and absorbance coefficients (e.g., molar extinction coefficients) over a wide range of the light spectrum, they are exceedingly fluorescent and thus are considered an excellent reagent for use in fluorescence applications, especially in flow cytometry (Table 4).

Phycoerythrin (PE), allophycocyanin (APC), and their tandem fluorophore conjugates are among the most suitable phycobiliproteins for flow cytometry applications. Conjugation of these dyes to macromolecules with biological specificities, such as antibodies, protein A or streptavidin, can be used in fluorescence-based detection applications that require high sensitivity but not necessarily photostability, such as fluorescence-activated cell sorting (FACS) and immunophenotyping. Compared with organic and synthetic fluorescent dyes, the advantages of phycobiliproteins as fluorescent labels include; long-wavelength fluorescence excitation and emission to minimize interference by auto-fluorescence from biological materials, minimal fluorescence quenching, high water-solubility, significant Stokes shifts with well-resolved emission spectra for multicolor analysis and multiple sites providing for stable conjugation with organic and synthetic compounds, including antibodies, cyanine dyes, iFluor™ dyes, and mFluor™ dyes.

Table 4. PE, APC, and tandem dyes and kits for labeling antibodies, proteins, and other biomolecules.

Phycobiliprotein Laser Ex max Em max Dye/Tandem¹ ReadiUse™ Dye/Tandem² Buccutite™ Antibody Labeling Kit
Phycoerythrin (PE) Blue/ Green/ Yellow 495, 546, 566 574 ABD-2558
ABD-2556
ABD-2557
ABD-2500
ABD-2501
ABD-1312
ABD-1310
PE-iFluor™ 594 Blue/ Green/ Yellow 495, 546, 566 606 ABD-2600 ABD-2584
PE-Texas Red Blue/ Green/ Yellow 495, 546, 566 615 ABD-2619 ABD-2583 ABD-1343
ABD-1318
PE-iFluor™ 610 Blue/ Green/ Yellow 495, 546, 566 625 ABD-2700
PE-iFluor™ 647 Blue/ Green/ Yellow 495, 546, 566 666 ABD-2702 ABD-2577
PE-Cy5 Blue/ Green/ Yellow 495, 546, 566 666 ABD-2610 ABD-2580 ABD-1340
ABD-1322
PE-Cy5.5 Blue/ Green/ Yellow 495, 546, 566 671 ABD-2613 ABD-2581 ABD-1341
ABD-1316
PE-iFluor™ 660 Blue/ Green/ Yellow 495, 546, 566 695 ABD-2602 ABD-2579
PE-iFluor™ 700 Blue/ Green/ Yellow 495, 546, 566 708 ABD-2614 ABD-2585
PE-iFluor™ 710 Blue/ Green/ Yellow 495, 546, 566 740 ABD-2615
PE-iFluor™ 750 Blue/ Green/ Yellow 495, 546, 566 778 ABD-2704 ABD-2578
PE-Cy7 Blue/ Green/ Yellow 495, 546, 566 778 ABD-2616 ABD-2582 ABD-1342
ABD-1317
Allophycocyanin (APC) Red 651 660 ABD-2554
ABD-2555
ABD-2561 ABD-1313
ABD-1311
Cross-linked Allophycocyanin (CL-APC) Red 651 660 ABD-2552
ABD-2549
ABD-2550
ABD-2551
ABD-2503
ABD-2504
APC-Cy5.5 Red 651 700 ABD-2622 ABD-2586 ABD-1350
ABD-1320
APC-XFD700 Red 651 707 ABD-2624
APC-iFluor™ 700 Red 651 710 ABD-2623 ABD-2570 ABD-1347
ABD-1319
APC-Cy7 Red 651 779 ABD-2625 ABD-2587 ABD-1351
ABD-1321
APC-XFD750 Red 651 782 ABD-2627
APC-iFluor™ 750 Red 651 793 ABD-2626 ABD-2571
APC-iFluor™ 800 Red 651 819 ABD-2630 ABD-2572

 

Tandem Dyes Further Expand Options for Multicolor Immunofluorescence Panels

When developing high-plex phenotypic panels, the use of spectrally similar fluorophores becomes practically unavoidable. One solution to this problem is to separate the complex fluorophore combinations by segregating them into cellular subpopulations that are gated and analyzed separately, thus limiting spectral spillover between populations. Another helpful approach for increasing panel size and diversity is by employing tandem dyes.

Tandem dyes comprise of two different fluorophores, a fluorescent donor and a longer-wavelength emitting fluorescence acceptor, that are conjugated to the same biomolecule. One fluorophore in the pair transmits energy to the other by the process of fluorescence resonance energy transfer (FRET). Since the second fluorophore emits light at a higher wavelength than that emitted by the first fluorophore, the number of fluorophores that can be distinguished using the same laser for excitation is effectively increased. Typically, PE and APC are used as donor fluorophores when generating tandem dyes, as illustrated with APC-iFluor™ 700 and PE-iFluor™ 750. In flow cytometry, tandem dyes are particularly suited for multicolor analysis of cells due to their exploitation of a single excitation source and their significantly large Stokes shifts.

PerCP (Peridinin-chlorophyll-protein complex), obtained from Dinophyceae spinosum, has an extremely high extinction coefficient, a high quantum efficiency, and a large Stokes shift. It is especially well excited by the 488 nm argon-ion laser, with its maximum emission peak at 677 nm. PerCP protein is commonly employed for fluorescent immunolabeling, especially fluorescent-activated cell sorting (FACS). Tandem conjugates with cyanine dyes, such as PerCP-Cy5.5, can be excited with a standard 488 nm laser and emits in the far red at a longer wavelength for multicolor flow cytometric analysis of cells. These multiple emission wavelengths make PerCP-cyanine conjugates potentially useful fluorochromes for multicolor analysis with FITC, PE, and other fluorophores. AAT Bioquest offers iFluor™ and mFluor™ protein labeling dyes, which are generally brighter and more photostable than the corresponding cyanine dyes of similar wavelengths.

24-Color panel for identifying circulating cell subsets in human peripheral blood

Specificity Violet Laser Fluorochromes Specificity Blue Laser Fluorochromes Specificity Red Laser Fluorochromes
CCR7 mFluor™ Violet 420 CD11c XFD488 (Alexa Fluor® 488 Equivalent) CD27 APC
CD19 mFluor™ UV420 CD45RA iFluor™ 488 CD123 iFluor™ 647
CD16 mFluor™ Violet 450 CD3 iFluor™ 532 CD127 APC-iFluor™ 700
TCR gamma delta mFluor™ Violet 500 CD25 PE HLA DR APC-iFluor™ 750
CD14 mFluor™ Violet 510 IgD PE-iFluor™ 594    
CD8 mFluor™ Violet 590 CD95 PE-Cy5    
CD1c mFluor™ Violet 610 CD11b PerCP-Cy5.5    
PD-1 Brilliant Violet 650™ CD38 PerCP-eFluor® 710    
CD56 Brilliant Violet 711™ CD57 PE-Cy7    
CD4 Brilliant Violet 750™        
CD28 Brilliant Violet 785™        
  1. Brilliant Violet™ is a trademark of Sirigen Group Ltd.
  2. eFluor® is a trademark of Thermo Fisher Scientific

 

Conclusion

The requirement to simultaneously interrogate increasingly more phenotypic features in complex cell populations, such as human peripheral blood cells, has demanded the implementation of more powerful instrumentation adaptations beyond those found in traditional flow cytometry. One important advance in multicolor immunofluorescence analysis has been the introduction of full spectrum multicolor spectral flow cytometry. This technology captures the entire emission spectrum of fluorophores using arrays of highly sensitive light detectors and has enabled simultaneous characterization of as many as 40 parameters in a single sample (Park et al., 2020). Implementation and broad adoption of this new flow cytometry-based instrumentation require careful design and validation of optimized multicolor immunofluorescence panels (OMIPs). These, in turn, require the availability of high-performance series of reactive organic fluorophores, fluorescent proteins, and tandem fluorophore-fluorescent protein conjugates that span the UV-visible-NIR region of the light spectrum.

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