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 Cytek 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.

Figure 3. Example of autofluorescence extraction. PrimeFlow RNA detection was used to label mRNA in human U937 cells. Cells were treated with PrimeFlow RNA detection kit and were either unstained or stained before detection on a 3-laser spectral flow analyzer* (Panel A). Unstained cells were mixed with stained cells and analyzed before and after autofluorescence removal (Panel B).


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 fluorsEmission max (nm)Fluorescent proteinsOther dyes
400–500Alexa Fluor 405421Azurite,
CFP,
Cerulean,
TagBFP,
mTurquoise,
AmCyan
Brilliant Violet 421,
Horizon V450,
VioBlue,
Horizon BV480
Super Bright 436436
eFluor 450450
Pacific Blue455
500–600Pacific Green500 Horizon V500,
Brilliant Violet 510,
VioGreen,
Brilliant Violet 570
eFluor 506510
Pacific Orange550
600–700Super Bright 600600 Brilliant Violet 605,
Brilliant Violet 650
Qdot 605605
Super Bright 645645
Qdot 655655
700–800Super Bright 702702 Brilliant Violet 711,
Horizon BB700,
Brilliant Violet 750,
Brilliant Violet 785,
Horizon BV786
Qdot 705705
Super Bright 780780
Qdot 800790
Emission range (nm)Recommended fluorsEmission max (nm)Fluorescent proteinsOther dyes
500–600Alexa Fluor 488520EGFP,
Emerald GFP,
mCitrine,
Venus,
EYFP,
RFP
Horizon BB515,
VioBright FITC,
Vio 515
Vio Bright 515
FITC520
Alexa Fluor 532550
PE576
600–700PE-eFluor 610607 PE-Dazzle 594,
Horizon BB700,
PE CF594
PE-Texas Red625
PE-Alexa Fluor 610630
PE-Cyanine5670
PerCP675
PE-Cyanine5.5690
PerCP-Cyanine5.5690
700–880PerCP-eFluor 710710 PerCP-Vio 710,
PE Vio770
PE-Alexa Fluor 700720
PE-Cyanine7780

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 Cytek 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 fluorophores 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
   
SB436eF450eF506 Pacific Orange SB600SB645SB702SB780FITCAF532PE PE-eF610 PE-Cyanine5 PE-Cyanine5.5 PerCP-eF710 PE-Cyanine7 APCAF647AF700 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
   
BV421SB436eF450BV480eF506Pacific OrangeBV570SB600SB645SB702BV750SB780FITCAF532PE PE-eF610 PE-Cyanine5 PE-Cyanine5.5 PerCP-eF710 PE-Cyanine7 APCAF647AF700 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.

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

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.

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