Three Tips for Assembling a Multicolor Flow Cytometry Panel

The widespread use of flow cytometry as an analytical tool has been instrumental in advancing research progress in the life sciences. With an ever-expanding repertoire of highly specific antibodies, fluorophores with unique spectral characteristics, and more versatile flow cytometry instruments, researchers are now able to construct multicolor flow panels that allow for simultaneous analysis of more than 20 parameters. But this increasing complexity often translates into higher technical difficulty. As more fluorophores are assembled into a panel together, it is important that their assignments to cellular markers are designated strategically, because a suboptimally designed panel will negatively impact experimental results. In this article, we’ll discuss some of the basic principles and considerations in constructing panels for your multicolor flow experiment.

It’s all about balance—fluorophore brightness vs. antigen density

Matching expected protein abundance and fluorophore brightness is one of the primary considerations in constructing a well-designed, balanced panel. The brightness of a fluorophore is commonly estimated based on staining index¹ (SI).BioLegend's Fluorophore Brightness Index provides relative indications of fluorescence intensity above the background for fluors commonly used in flow cytometry. It is important to note that analysis of low-abundance cell markers using bright fluorophores maximizes the resolution of this population. Also, a panel designed using the opposite criteria (i.e., dimly expressed markers on dim fluors, highly expressed markers on bright fluors) will likely lead to several downstream issues, including voltage imbalance and amplified spreading error².

The best resource pertaining to cell marker density and cell population frequency in a given sample would likely come from previous characterization, such as published
data^4–6. Antibody manufacturers can also provide valuable information. All flow cytometry antibodies sold by BioLegend include representative data figures on the product datasheet. Browsing through some of the presented data across different conjugates may help you gauge expected expression levels for your markers of interest, and determine which fluorophore would be a good fit for your experiment.

Minimizing spectral overlap

A careful selection of an antibody–fluorophore combination will also minimize spectral overlap and spillover. You should avoid using fluors with known significant spillover to detect co-expressed markers, as well as those that you are planning to directly compare in a bivariate plot. Having a preplanned gating and analysis strategy for your experiment is essential here. Comparing excitation/emission properties of different fluors on BioLegend’s Fluorescence Spectra Analyzer can help you choose the best fluorophore combination for your markers of interest. This interactive tool can be used to analyze excitation and emission spectra of commonly used fluorochromes for flow cytometry.

The unexpected result—biological and technical variability

Even after optimizing an antibody–fluorophore combination for a panel on paper, your flow staining results may vary depending on experimental context. Here are some biological and technical questions to consider for further optimization:

• Sample type. Are you working with stimulated/diseased samples that may be affecting marker expression compared to healthy, unstimulated controls?
• Sample preparation. Are the samples enzymatically digested, which can potentially affect surface marker integrity³?
• Antibody titration. Did you define the optimal antibody usage amount based on titration to maximize signal-to-noise ratio?
• Instrument and software-related factors. Are the flow cytometer settings, such as acquisition gain/voltage and compensation, adjusted accordingly using proper controls?

BioLegend offers a wide variety of useful online resources that can help answer such questions regarding panel design, including information about cell markers and flow cytometry controls. Our technical support team can also assist with panel building—a service offered free-of-charge to anyone interested in performing multicolor panel experiments. Also helpful is the Multicolor Panel Selector tool, focused on choosing the correct flurophores for your experiments based on your instrument setup and markers of interest.

Concluding remarks

Constructing a balanced, well-strategized panel for your multicolor flow cytometry experiment is an important first step for obtaining successful results. In addition to a well-designed panel, other factors not detailed here include instrumentation, software, and experimental and technical controls—all essential in ensuring that you obtain good data.

Related products

Brilliant Violet™ Antibody Conjugates

PE/Dazzle™ 594 Conjugates

Flex-T™ MHC Tetramers

APC/Fire™ 750 Conjugates

Veri-Cells™ Lyophilized Control Cells for Flow Cytometry

Zombie Dyes for Live Cell/Dead Cell Discrimination

References

1. Maecker, H.T. et al. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A. 2004 Dec;62(2):169-73

2. Nguyen, R. et al. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry A. 2013 Mar;83(3):306-15

3. Autengruber, A. et al. Impact of enzymatic tissue disintegration on the level of surface molecule expression and immune cell function. Eur J Microbiol Immunol. 2012 Jun; 2(2): 112–120

4. Ginaldi, L. et al. Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry. J Clin Pathol. 1996 Jul;49(7):539-44

5. Unternaehrer, J.J. et al. The tetraspanin CD9 mediates lateral association of MHC class II molecules on the dendritic cell surface. Proc Natl Acad Sci U S A. 2007 Jan 2;104(1):234-9

6. Antal-Szalmas, P. et al. Quantitation of surface CD14 on human monocytes and neutrophils. J Leukoc Biol. 1997 Jun;61(6):721-8