Regular advances in reagents and instrumentation for flow cytometry allow researchers to run increasingly complex multicolor experiments. This increased complexity requires a more strategic approach to experimental design, especially when choosing which fluorophore to pair with each antibody. With dozens of fluorophores to choose from, it’s easy to find one that excites and emits in any segment of the spectrum.

The current study evaluates the performance of 25 fluorophores, including organic dyes and recently developed Qdot® nanocrystals, each conjugated to an anti-CD4 antibody. The relative brightness of each conjugate was calculated using the standard stain index. The data indicate that some dyes have a low stain index and should not be used with low-abundance markers. However, we demonstrate that even a dim dye can be an effective part of a panel, as long as it is carefully matched to the expression of the antigen being detected. Understanding the relative brightness of commonly used dyes and knowledge of antigen expression are crucial elements of experimental design to ensure accuracy of results.

Matching Dyes to Appropriate Markers

Some cellular antigens are very highly expressed and can be used with a variety of fluorophores, even those with relatively low brightness. However, many antigens have a low expression density and must be paired with a very bright fluorophore to differentiate between positive and negative populations. An example of appropriate matching is shown in Figure 1. A bright fluorophore such as allophycocyanin(APC) is needed to distinguish between weakly positive and negative populations. Relatively dim dyes, such as Pacific Orange™, should be reserved for highly expressing markers.

Matching fluorophore brightness to marker expression level.

Figure 1. Matching fluorophore brightness to marker expression level. Human peripheral blood mononuclear cells were stained with APC, Alexa Fluor® 488, or Pacific Orange™ conjugates of mouse anti–human CD4 antibody. (Top Row) Histograms produced by collecting 10,000 events in a gate on monocytes, which have low expression of CD4. In this case, positive and negative populations cannot be distinguished by the relatively dim Pacific Orange™ dye. (Bottom Row) Same data set but with a gate on lymphocytes, which have high cell surface expression of CD4, so all fluorophores can be used to distinguish positive and negative populations.

Tips for Fluorophore Selection

Substantial variation in brightness can be observed between different fluorophores conjugated to the same antibody (Figure 2).

Table 1 lists common anti-CD4 fluorophore conjugates and their properties in order of decreasing brightness based on the stain index. In addition to the stain index, other approaches such as signal-to-noise ratio can be used to assign relative brightness. Actual fluorophore brightness depends on many factors, including the laser power, emission filter, and instrument on which they are used.

In addition to fluorophore brightness, when designing a flow cytometry experiment, keep a few guidelines in mind. Know the configuration of the instrument being used (laser and filters). Select fluorophores compatible with your instrument’s laser and filters. Use a tool like Invitrogen’s Spectra Viewer during the dye selection process to understand the spectral overlap of fluorophores (see sidebar). Always protect vials of fluorophores and stained samples from light to avoid light-mediated degradation and photobleaching. This is especially important with the use of tandem conjugates. Finally, validate that your fixation and permeabilization method is compatible with your chosen antibody–fluorophore conjugates (Figure 3).

Comparison of fluorescent signals from relatively dim, average, and bright fluorophores.

Figure 2. Comparison of fluorescent signals from relatively dim, average, and bright fluorophores. Lysed whole blood was stained with mouse anti–human CD4 conjugated to Pacific Orange™ (Left), Alexa Fluor® 700 (Center), or allophycocyanin (Right). The left-hand peak shows autofluorescing cells, the right-hand peak, CD4+ cells. Cells were analyzed on a BD™ LSR II flow cytometer with FACSDiva® version 6.1 software. Each histogram represents 10,000 cells collected in a lymphocyte gate.

Table 1. Properties of common mouse anti–human CD4 fluorophore conjugates.

Fluorophore Antibody Clone Excitation Max (nm) Exmission Max (nm) Exmission Filter Stain index*
APC-Cy® 5.5 tandemS3.5650690710/50108.97
PE-Cy®5.5 tandemS3.5496,565690695/40105.91
Alexa Fluor® 488 dyeS3.5495519525/5091.72
Alexa Fluor® 647 dyeRPA-T4650668660/2074.35
PE–Alexa Fluor® 610 tandemS3.5428628620/1070.71
FITC dyeS3.5493525525/5056.40
PE-Cy® 7 tandemS3.5496,565774780/6053.70
PE–Alexa Fluor® 700 tandemS3.5496,565723720/3052.45
PE–Texas Red® tandemS3.5496,565613695/4040.85
APC-Cy® 7 tandemRPA-T4650774780/6035.81
Qdot® 605 nanocrystalS3.5350605605/2035.17
APC–Alexa Fluor® 750 tandemS3.5645775780/6031.91
Alexa Fluor® 700 dye S3.5696719710/5024.85
PerCP-Cy® 5.5 tandemLeu-3a482690710/2021.93
Qdot® 655 nanocrystalS3.5350655655/2020.62
Qdot® 705 nanocrystalS3.5350720720/2018.38
APC-H7 tandemRPA-T4650774780/6015.95
Pacific Blue™ dyeS3.5410455450/5014.61
Alexa Fluor® 405 dyeS3.5401421450/5010.01
PerCP complexS3.5482675695/408.75
Pacific Orange™ dyeS3.5400551585/426.06
*Calculated by subtracting the mean fluorescence intensity of the negative peak from the positive peak and dividing by twice the standard deviation of the negative peak. Sufficient concentrations of mouse anti–human CD4 conjugates were used to produce complete binding saturation.
Ensuring that conjugates are not affected by cell processing.
 Figure 3. Ensuring that conjugates are not affected by cell processing. Human lymphocytes were stained with anti-CD4–Qdot® 655 conjugate for 20 min at room temperature (RT). Stained cells were then subjected to various treatments to evaluate the stability of the conjugate: (A) no further treatment; (B) fixation with Invitrogen™ IC Fixation Buffer for 20 min at room temperature; (C) treatment for 20 min at RT with Invitrogen™ IC Fixation Buffer, 20 min at RT with 1% saponin in phosphate-buffered saline (PBS); (D) treatment with FIX & PERM® Cell Permeabilization Reagents: 10 min at RT with FIX & PERM® Fixation Medium, 20 min at RT with FIX & PERM® Permeabilization Medium. All treatments included intermediate and final wash steps. These plots show very little change in signal after the various treatments, indicating that this conjugate is not affected by cell processing.

Improving Your Strategy

Complex flow cytometry experiments require a strategic approach to experimental design. Antibodies and fluorophores each have multiple properties that must be optimized to achieve good experimental results. In this study, we evaluated the importance of matching each antibody to a dye of appropriate brightness. Even a dim dye can be an effective part of a panel, as long as it is paired with a highly expressed antigen.
