One of the most important considerations when setting up a flow cytometry experiment is deciding which fluorophores to use. But with flow cytometry seeing increased utility to detect multiple targets in parallel, this has become far more complex than simply identifying fluorophores that best fit the instrument configuration. Here, we look into why developing an antibody panel can make fluorophore selection challenging and provide some tips to help you choose the right fluorophores for your flow cytometry experiment.
When selecting fluorophores for flow cytometry, it is of paramount importance to understand the configuration of your flow cytometer. This means knowing the number and types of lasers and filters it is equipped with since these will determine which fluorophores are compatible for use. Lasers should emit light close to the maximum excitation wavelength (λex) of the chosen fluorophore, while filters should be suitable to detect light near the maximum emission wavelength (λem) without registering light from other sources. Where a panel of fluorophores will be used, it is recommended to distribute fluorophores as widely as possible across lasers and filters to reduce interference.
To avoid spill-over that can impact resolution and sensitivity, it is good practice to pair bright fluorophores (like PE or APC) with low abundance cellular targets and dim fluorophores (like Alexa Fluor 700 or PerCP) with highly expressed proteins. This improves the ability of the flow cytometer to discriminate between specific signal and background fluorescence arising from variables such as non-specific staining and cellular autofluorescence. The brightness of a fluorophore is defined by its extinction coefficient (the capacity to absorb light at a given wavelength, measured in M−1 cm−1) and its quantum yield (the number of photons emitted per absorbed photon, a value ranging from 0–1), with higher values being indicative of brighter emissions. Where an antibody is not available bound to the desired fluorophore, many antibody suppliers offer a custom antibody labeling service for a nominal fee.
Every fluorophore has characteristic excitation and emission spectra, defined by λex, λem, and a Stokes shift (the difference between λex and λem). Where a panel of antibodies will be run simultaneously, these factors should be reviewed with the aim of minimizing spectral overlap and reducing the need for compensation. Online tools for viewing fluorophore spectra are widely available and can help to inform panel design. Fluorophore photostability, as well as the stability of fluorophores after fixation, are also important considerations depending on the experimental process.
Table: Typical dyes excited by violet lasers. For a more complete listing of fluorochromes click here.
Where the use of spectrally similar fluorophores is unavoidable, it is advised that these are paired to cellular sub-populations that will be gated and analyzed separately as this can limit the spectral spillover between populations. However, an alternative approach is to increase panel size and diversity by using tandem dyes. These specialized reagents consist of two different fluorophores that can be conjugated to the same antibody, whereby one fluorophore transmits energy to the other by fluorescence resonance energy transfer (FRET). Because the second fluorophore emits light at a higher wavelength than that emitted by the first fluorophore, the number of readouts that can be obtained using the same laser for excitation is increased. PE and APC are widely used as donor fluorophores when generating tandem dyes, as seen with PE-Cy5.5 and APC-Cy7.
Large flow cytometry panels often have spectral spillover that must be accounted for. Calibration beads are a quick and easy tool for evaluating spectral spillover, as well as for assessing flow cytometer performance in general. Among the most widely used are rainbow beads, comprising populations of particles dyed to different fluorescent intensities, where each individual bead contains a mixture of fluorophores that are excited at wavelengths ranging from 365–650 nm. Other compensation bead types are specifically designed to capture the exact fluorescent antibody used in an experiment to best allow for spectral compensation in any panel. These beads can create specific positive and negative populations and allow for rapid pre-setup compensation as well as post-experimental compensation adjustments.
Image: RayBright® fluorochrome-conjugated antibodies with 50 µl of RayBright® Universal Compensation beads
Although the need for compensation can be avoided by using fluorophores with distinct, non-overlapping emission spectra, this quickly becomes impractical as panel size increases. Compensation controls are therefore critical to ensure any detected light is attributed to the correct fluorophore. Preparing compensation controls involves setting up a positive and a negative population for each individual fluorophore, where ideally both populations consist of the same cell type. Where this is not possible, rainbow beads or compensation beads, as mentioned above, are a viable alternative. It is important to note that color compensation using tandem dyes can be more challenging due to variations in dye conjugation; using the same batch of conjugated antibody is suggested for greater experimental consistency.
RayBiotech is a leading pioneer in the proteomics field and offers a comprehensive range of flow cytometry antibodies, RayBright® dyes, and flow cytometry dye labeling and testing services. To learn more, visit raybiotech.com