Full spectral analysis

Advances in flow cytometry instrumentation, as with spectral flow cytometers, coupled to a growing number of fluorescent labels and readouts have expanded applications and capabilities beyond conventional flow cytometry. Now with spectral flow cytometry analysis, researchers and scientists can investigate an increasing number of molecules of interest. Importantly, larger polychromatic panels still require careful selection of available fluorophores, fluorescent reagents and proteins. Here we provide spectral flow cytometry users a guide to selection of available fluorophores, assays and reagents that fit the needs of multiplexed spectral flow cytometry approaches in cell biology, immunology, cancer biology, microbiology, and plant biology.

Fluorescent combinations for spectral flow cytometry analysis

As the flow cytometry research community strives to increase panel complexity, different enabling technologies are becoming available to meet their scientific needs. Researchers are starting to turn to spectral flow cytometry analyzers, such as the Aurora™ spectral flow cytometer provided by Cytek Biosciences or the SA3800 provided by Sony Biotechnology. Spectral flow cytometers exploit the inherent emission pattern of each fluorescent molecule to generate a unique spectral signature (comparative examples in Figures 13). However, each instrument has different laser configurations and optical sensitivity, which is critical to understand when designing multicolor panels and choosing the appropriate fluorochrome combinations. In any case, individual fluorescent reference controls are needed, similar to that of single-color controls in conventional cytometry, in order to deconvolute or unmix the spectral signatures in polychromatic panels. Therefore, spectral flow cytometry analysis relies on the discrimination of unique spectral signatures rather than specific emission channels for detection, enabling the compatibility and distinction of many fluorescent combinations that were previously difficult or impossible to separate, as shown by PerCP and PerCP-eFluor 710 (Figure 1).

Note: Each spectral signature (Figures 1-3) generated displays the distribution of events as a function of intensity across the whole spectrum, for each laser. Interpreting the color scheme for each channel is similar to that of a heat map or density plot. Red represents the intensity at which most events had fluorescence, while yellow, green and blue represent decreasing numbers of events. Each laser’s set of detectors can have additional emission captured enabling the distinction between fluorophores, as indicated by the red boxes. However, based on this information dyes that emit at similar wavelengths can introduce spread into the other. Refer to Tools to achieve effective panel design for information on the amount of spread introduced among fluorophores.

A

Overlay of PerCP and PerCP-Cy5.5

B

Spectral analysis of fluorescent protein molecule and its tandem

Figure 1. Comparison of a large protein molecule and its tandem. (A). Overlay of PerCP and PerCP-eFluor 710 excitation and emission profiles, demonstrating their significant overlap, taken from Fluorescence SpectraViewer. (B). PerCP positive cells (top graph) compared to PerCP-eFluor 710 positive cells (bottom graph) analyzed on a 3-laser spectral flow cytometer* system. Although emission values are very similar, the unique patterns in the far-red channels allow for the two molecules to be discriminated.

Various classes of fluorescent probes and reagents can be multiplexed more readily in spectral flow cytometry experiments. For instance, In larger immunophenotyping panels when available labeling reagents become limited, the Invitrogen Alexa Fluor 647 dye can now be used in combination with larger fluorescent proteins such as with APC (Figure 2). When expanding panels to identify sub lineage cell types and associated markers, the violet laser-excitable Super Bright antibody conjugates can be taken advantage of in combination with traditional dyes like eFluor 450 and Pacific Orange. Flow cytometry antibody panels are the basis for immunophenotyping experiments, but inclusion of functional reagents such as CellTrace can be challenging in larger panels on conventional instrumentation. Spectral flow cytometers can allow for simpler integration of functional reagents by utilizing their unique spectral signatures. Our Invitrogen portfolio of fluorescent reagents and assays are the perfect complement to the burgeoning field of spectral cytometry analysis that can enable cell biologists, immunologists, and cancer biologists the ability to uncover deeper biological insights.

Spectral flow analysis of fluorescent molecules

Figure 2. Example of two compatible fluorochromes. Allophycocyanin (APC) (top graph) and Alexa Fluor 647 (bottom graph), are now compatible when analyzed on a 3-laser spectral flow cytometer*. Although emission profiles are similar, their unique patterns highlighted in the violet and blue channels allow for the molecules to be discriminated.

Invitrogen fluorescent probes are capable of elucidating biological mechanisms such as cell cycle stages, important cell signaling pathways, cell death through apoptosis, RNA detection, etc. Combining these probes to antibody staining requires careful calibration and the appropriate biological controls. As cellular autofluorescence can interfere with fluorescence detection in some protocols, spectral flow cytometry is capable of identifying and removing autofluorescence during analysis (see Figure 3 for an Invitrogen PrimeFlow RNA example). During spectral unmixing autofluorescence can be removed in order to better resolve and identify the true signal from the fluorescent probe of interest. This capability further enables combinatorial approaches of antibody labeling with fluorescent-based functional probes to elucidate the biological significance during spectral flow cytometry experiments.


Find compatible reagents for a 3-laser spectral flow cytometer

Many Invitrogen reagents can be combined in your spectral flow cytometry experiment. Use the selection guide below to find available fluorescent antibody label options and functional reagents for a spatially offset 3-laser spectral flow analyzer*. Many of the listed fluorophores can be used in combination, however there are still some incompatibilities. Please refer to Tools to achieve effective panel design to help guide fluorophore selection when designing multicolor panels.

Selection guide for spectral flow cytometry analyzers

Emission range (nm) Recommended fluors Emission max (nm) Fluorescent proteins Other dyes
400–500 Alexa Fluor 405 421 Azurite,
CFP,
Cerulean,
TagBFP,
mTurquoise,
AmCyan
Brilliant Violet 421,
Horizon V450,
VioBlue,
Horizon BV480
Super Bright 436 436
eFluor 450 450
Pacific Blue 455
500–600 Pacific Green 500   Horizon V500,
Brilliant Violet 510,
VioGreen,
Brilliant Violet 570
eFluor 506 510
Pacific Orange 550
600–700 Super Bright 600 600   Brilliant Violet 605,
Brilliant Violet 650
Qdot 605 605
Super Bright 645 645
Qdot 655 655
700–800 Super Bright 702 702   Brilliant Violet 711,
Horizon BB700,
Brilliant Violet 750,
Brilliant Violet 785,
Horizon BV786
Qdot 705 705
Super Bright 780 780
Qdot 800 790
Emission range (nm) Recommended fluors Emission max (nm) Fluorescent proteins Other dyes
500–600 Alexa Fluor 488 520 EGFP,
Emerald GFP,
mCitrine,
Venus,
EYFP,
RFP
Horizon BB515,
VioBright FITC,
Vio 515
Vio Bright 515
FITC 520
Alexa Fluor 532 550
PE 576
600–700 PE-eFluor 610 607   PE-Dazzle 594,
Horizon BB700,
PE CF594
PE-Texas Red 625
PE-Alexa Fluor 610 630
PE-Cyanine5 670
PerCP 675
PE-Cyanine5.5 690
PerCP-Cyanine5.5 690
700–880 PerCP-eFluor 710 710   PerCP-Vio 710,
PE Vio770
PE-Alexa Fluor 700 720
PE-Cyanine7 780

Tools to achieve effective panel design

Like traditional flow cytometry, spectral flow cytometry enables live cellular analysis, which is critical when investigating numerous immunological areas. For instance, when interrogating the tumor micro-environment, flow cytometric assays are useful approaches to cytokine profiling, assessing tumor specific antigens, immune checkpoint discovery, CAR T cell therapy, among others. Careful panel design for spectral flow cytometry analysis requires an understanding of an instrument’s capabilities, cell lineage sub populations, their expected antigen density, and the available antibody-conjugates and their properties. The information provided in the following tables is intended to guide researchers in their selection of fluorochromes for polychromatic panels when using the Aurora 3-laser system.

Table 1. Spread matrix of 20 Invitrogen fluorophores that can be used simultaneously in spectral flow cytometry. A fluorescent dye matrix that demonstrates the level of spread among dyes. Fluorophores of each row impact the spread of the fluorophore in the column. Although all dyes in the matrix can be used together, the darker red shading means one fluorophore has increased spread into the other, and needs closer attention when designing panels and interpreting data.

Click on each cell to see comparisons of spectral signatures and the percent reduction of the dye's cross-staining index. The comparison of spectral signatures is a good indicator of the impact that one fluorophore can have on the other, while the percent reduction of the dye's cross-staining index is intended to be used as a quantitative measurement of resolution. By understanding the influence of one fluorophore's spread (row) into the resolution of the other (column), the selection of appropriate fluorophores that can be used in combination is much simpler. In this matrix all fluororphores were controlled for the same antigen (CD4), and is intended to be a valuable point of reference in panel design.







Click on a cell


to see spectral


signature


comparison
     
SB436 eF450 eF506 Pacific Orange SB600 SB645 SB702 SB780 FITC AF532 PE PE-eF610 PE-Cyanine5 PE-Cyanine5.5 PerCP-eF710 PE-Cyanine7 APC AF647 AF700 APC-eF780
  SB436                                        
eF450                                        
eF506                                        
Pacific Orange                                        
SB600                                        
SB645                                        
SB702                                        
SB780                                        
  FITC                                        
AF532                                        
PE                                        
PE-eF610                                        
PE-Cyanine5                                        
PE-Cyanine5.5                                        
PerCP-eF710                                        
PE-Cyanine7                                        
  APC                                        
AF647                                        
AF700                                        
APC-eF780                                        

Table 2. Spread matrix of 24 fluorophores that can be used simultaneously in spectral flow cytometry. When expanding the panel with additional fluorophores refer to the fluorescent dye matrix below, which demonstrates the level of spread among dyes. Fluorophores of each row impact the spread of the fluorophore in the column. Although all dyes in the matrix can be used together, the darker red shading means one fluorophore has increased spread into the other, and needs closer attention when designing panels and interpreting data.







Click on a cell


to see spectral


signature


comparison
     
BV421 SB436 eF450 BV480 eF506 Pacific Orange BV570 SB600 SB645 SB702 BV750 SB780 FITC AF532 PE PE-eF610 PE-Cyanine5 PE-Cyanine5.5 PerCP-eF710 PE-Cyanine7 APC AF647 AF700 APC-eF780
  BV421                                                
SB436                                                
eF450                                                
BV480                                                
eF506                                                
Pacific Orange                                                
BV570                                                
SB600                                                
SB645                                                
SB702                                                
BV750                                                
SB780                                                
  FITC                                                
AF532                                                
PE                                                
PE-eF610                                                
PE-Cyanine5                                                
PE-Cyanine5.5                                                
PerCP-eF710                                                
PE-Cyanine7                                                
  APC                                                
AF647                                                
AF700                                                
APC-eF780                                                

Table 3. Invitrogen reagents that are not recommended to be used together in spectral flow cytometry. All fluorophores in the spread matrices above are usable together. However, based on the needs of the experiment and the available antibody conjugates, adjustments might need to be made. Refer to the table below as a guide when customizing your panel. Each cell contains a list of fluorochromes and viability dyes that are not recommended to be used together.

Staining Indices (SI) of 53 fluorophores

Figure 4. Fluorophore staining index (SI) comparison for spectral flow cytometry. PBMCs were isolated from whole blood and analyzed on a 3-laser spectral flow analyzer*. Staining was performed with CD4 primary antibody and subsequently labeled with secondary fluorophore. All antibodies were titered and ranked based on decreasing SI value. Stain indexes may vary from instrument to instrument, both in terms of absolute value and relationship to other fluorochromes.

For technical assistance, please email flowsupport@thermofisher.com. Alternatively, if you’re located within the US or Canada, you can call 800 955 6288, press 8, then enter extension 59797.

* All spectral flow cytometry data shown were generated by Cytek Biosciences on a Cytek® Aurora™ spectral flow cyometer 3-laser system and analyzed using SpectroFlo™ software.

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