Hundreds of fluorochromes are available to users conducting flow cytometry assays, each with unique properties that influence panel resolution and data quality. Selecting the right combination can be overwhelming, leading many researchers to rely on familiar choices that may introduce spillover and reduce clarity.
It’s essential to optimize panel design to minimize data loss and improve resolution. This guide simplifies fluorochrome selection by ranking them based on spillover and resolution, helping users design cleaner, more effective panels.
Download this guide to discover:
- How fluorochrome spillover impacts resolution and data interpretation
- Strategies for selecting fluorochromes based on antigen density and expression
- A performance chart to simplify panel design and enhance data quality
Fluorochrome
Performance Guide
Flow cytometry users choose from hundreds of fluorochromes for their conventional and spectral flow cytometry assays. The
physical properties of all fluorochromes are not the same, and differences in resolution and spillover can significantly impact
panel resolution and data interpretation. The process of learning every fluorochrome’s properties can seem overwhelming
and intimidating. As a result, flow cytometry users feel more comfortable using familiar fluorochromes, such as PerCP-Cy5.5
or PE tandems, which may present challenges and even limit or compromise the quality of data.
This guide is intended to help simplify panel design and minimize loss of data quality and resolution. By using the
Fluorochrome Performance Chart and the Fluorochrome and Antigen Pairing Guide presented here, you can easily prioritize
fluorochromes with minimal spillover and appropriate resolution.
Chart contains representative fluorochromes compatible with a 5-laser spectral flow cytometer. Table may differ based on instrument configuration and settings. Spillover
ranking is based on cross-laser excitation and does not take into account spillover into adjacent detectors.
Prioritize clean fluorochromes and simplify panel design
Fluorochrome Performance ChartFigure 1
Generating the Fluorochrome
Performance Chart
The Fluorochrome Performance Chart organizes and ranks fluorochromes based on spillover and
resolution, two of the most critical factors in fluorochrome selection.
Fluorescence spillover defines the spectral overlap between the emission profile of two
fluorochromes. Spectral overlap can be managed through compensation or spectral unmixing
to prevent data artifacts. However, these two processes do not eliminate spillover spread, the
main source of background and loss of resolution in multiparameter flow cytometry assays.
Spread is directly correlated with spillover (the level to which two fluorochrome profiles
overlap) and signal intensity (antigen density and fluorochrome brightness).
Spillover is evaluated and ranked based on the analysis of a given fluorochrome’s full emission profile
across five lasers. Fluorochromes with a single emission peak are ranked as 1 and fluorochromes excited
by multiple lasers are ranked as 2, 3 or 4 (additional peaks were counted if the spillover value was greater
than 15% of the main peak signal). Adjacent spillover is not taken into consideration for this ranking.
Resolution is determined by comparing the stain index of fluorochromes conjugated to several antibody
clones on a variety of flow cytometers to capture variation in configurations. A ranking of 1 identifies dim
fluorochromes with relatively low stain index, and 4 identifies brighter fluorochromes with higher stain
index. Scan the QR code for a list of fluorochrome resolution rankings by primary excitation laser line.
By prioritizing fluorochromes in columns 1 and 2, users can design panels while minimizing resolution loss
due to spillover-spreading error (spread). When additional challenges are present, such as limited reagent
availability or designing very large panels, the other fluorochromes (columns 3 and 4) can be carefully
incorporated into the panel.
Fluorochrome resolution defines the degree of separation between the negative and
positive populations. Signal intensity also contributes to the total amount of spread, where
cells expressing antigens at higher density will introduce higher spread.
Relative Fluorochrome
Resolution Chart
Detector
Normalized Emission
RB545
Detector
Normalized Emission
PerCP-Cy5.5
Detector
Normalized Emission
PE
Detector
Normalized Emission
BV605
Pairing clean fluorochromes
and markers
While the Fluorochrome Performance Chart provides guidance for the prioritization of fluorochromes with
minimal impact to resolution, fundamental panel design principles then need to be followed to build a panel.
The Fluorochrome and Antigen Paring Guide provides recommendations for the appropriate use of
fluorochromes based on target antigen profile and density. Depending on the panel markers and instrument
configuration, the total number of minimally overlapping fluorochromes that may be used together will vary.
Clearly resolved
High
Use dim fluorochromes
with minimal spillover
BUV395
BUV496
BUV805
V4504
V500 or BV5105
BV750
BV786
FITC or AF4881
RB545
AF7002
APC-H7
Not clearly resolved
Low/Medium
Use bright
fluorochromes
BUV615
BV4214
BV4805
BB5151
RB613
RB705
RB744
RB780
PE3
RY5863
RY610
RY703
RY775
APC or AF647
R7182
Variable
Low-to-high/Unknown
Use bright fluorochromes
with lowest spillover
BV4214
BB515
RB744
RB780
RY586
Antigen profile
Antigen density
Recommended
fluorochromes
Fluorochrome and Antigen Pairing Guide
Note: Fluorochromes with a single emission peak may still impact resolution of other neighboring fluorochromes with an adjacent main emission peak (e.g., RY586
and RY610, BB515 and RB545, RB744 and RB780). If possible, avoid pairing these adjacent fluorochromes with co-expressed markers with high antigen density.
For “Clearly Resolved” and highly expressed markers, resolution is minimally impacted by the spillover spread that may be
introduced by fluorochromes with adjacent main emission peaks (e.g., BB515 and RB545), especially if the two markers are not
co-expressed. “Not Clearly Resolved Markers” are less likely to introduce spread due to low antigen density. For variable markers
and markers with unknown expression levels, bright fluorochromes with minimal spillover will help ensure resolution of the
populations at the low end of expression range, while minimizing any spread from the population at the high end of expression
range.
Note that although feasible in spectral flow cytometry, use of very similar fluorochromes in a panel (e.g., FITC and BB515, APC
and Alexa FluorTM 647) should be avoided to prevent high spread.
Count
Count
Count
CD3 RB545 PD-1 RB780 CD45RA BB515
1 Use either FITC, Alexa FluorTM 488 or BB515
in the same panel
2 Use either R718, Alexa FluorTM 700 or
APC-R700 in the same panel
3 Use either RY586 or PE in the same panel
4 V450 and BV421 can be used together in
spectral flow cytometry with minimal
resolution impact
5 BV480 and either BV510 or V500 can be
used together in spectral flow cytometry with
minimal resolution impactPutting the Performance Guide to use
A 17-color flow cytometry panel was designed following the strategy provided in this guide. The list of usable fluorochromes was first
narrowed down based on low spillover ranking from the Fluorochrome Performance Chart (Figure 1, columns 1 and 2). Fluorochromes
were then selected and assigned to markers based on antigen profile, expression profile and reagent availability, as per the Fluorochrome
and Antigen Pairing Guide.
The use of overall clean dyes with minimal spillover ensured the clear resolution of several lymphocyte populations and the analysis of
inhibitory receptors’ expression therein.
The continuous development of fluorochromes with lower cross-laser excitation offers more and new options for the design of flow
cytometry panels with reduced spread and higher biological resolution. Combine the information from the Fluorochrome Performance
Guide and the Antigen Pairing Guide to simplify the design of high-quality flow cytometry panels.
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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Marker
CD45
CD3
CD4
CD8
CD56
CD16
CD45RA
CD197
CD95
PD-1
TIGIT
KLRG1
TCR GD
CD25
FoxP3
CD19
CD127
Fluorochrome
BUV395
RB545
APC-H7
BUV805
BV480
RY610
BB515
BUV615
BV421
RB780
AF647
R718
RB744
RB705
RB613
BV786
RY586
1. CD45+ non -B cells
2. CD3- cells
3. CD3+ cells
4. CD56bright cells
5. CD56dimCD16+ cells
6. NKT-like cells
7. TCRγδ + T cells
8. Conventional T cells
9. TEMRA
10. CD197+CD45RA+ cells
11. Tcm
12. Tem
13. Conventional CD4+ T cells
14. CD25highCD127low cells
Representative analysis of human PBMCs isolated from a healthy donor and stained with the panel shown on the left. Several lymphocyte populations, including B cells, NK cells
and T cells (gamma delta, Tregs, memory CD4+ and CD8+ cells) could be clearly resolved. The bottom insert shows a representative analysis of the expression of TIGIT and PD-1
throughout distinct subsets of naïve and memory CD8+ T cells. Samples were acquired and spectrally unmixed on a BD FACSCSymphonyTM A5 SE Flow Cytometer.
Singlets CD45+ non-B cells CD3- cells CD3+ cells
B cells
1
2
3
4
5
6 7
8
Conventional T cells
CD4+ T cells
CD8+ T cells
CD4+ T cells CD25hiCD127low
Tregs
CD4+ T cells
9 10
12 11
Tcm Tem TEMRA
CD8+CD197+CD45RA+
Tscm
Naïve
CD8+ T cells
9 10
12 11
13 14
Naïve Tscm
CD8+ T cells
CD8+ T cells CD8+ T cells
CD19 BV786
CD56 BV480
CD3 RB545
CD56 BV480
CD16 RY610
CD56 BV480
TCRyō RB744
CD4 APC-H7
CD8 BUV805
CD45RA BB515
CD197 BUV615
CD127 RY586
CD25 RB705
FoxP3 RB613
CD25 RB705
CD127 RY586
TIGIT AF647
PD-1 RB780
TIGIT AF647
PD-1 RB780
TIGIT AF647
PD-1 RB780
TIGIT AF647
PD-1 RB780
TIGIT AF647
PD-1 RB780
TIGIT AF647
PD-1 RB780
CD45RA BB515
CD197 BUV615
CD95 BV421
CD197 BUV615
CD45 BUV396
KLRG1 R718