As flow cytometry systems continue to offer greater capabilities in multiparameter detection—with new cytometers capable of accommodating upwards of 64 fluorescence channels—the ability to study many aspects of cell biology becomes limited only by creativity.
Researchers now have straightforward access to countless antigen-specific antibodies combined with a diverse selection of fluorophores.
A multicolor flow cytometry panel that is carefully designed can be crucial in answering many complex cellular questions, including those centered on cellular phenotypes, cell populations, cell-cell interactions, chemical responses, and rare events.
However, as more antibodies and fluorochromes are included within a given panel, careful planning is recommended to ensure robust resolution, sensitivity, and biological accuracy.
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Some important considerations in building a multicolor flow cytometry panel:
- Fluorophore spectra - Fluorophore excitation and emission spectra should be reviewed with the aim of minimizing spectral overlap, which refers to unexpected spillover of fluorescence into a detector channel. This causes noise that will need to be corrected via compensation.
- Target abundance and brightness - The abundance of a given cellular target should be taken into account, as it can influence sensitivity. It is a good idea to pair bright fluorophores with low abundance antigens and dim fluorophores with highly expressed proteins.
- Antibody types - Various antibody types may factor into a panel decision. For example, monoclonal antibodies are less likely to exhibit off-target cross-reactivity than polyclonals. Certain IgG isotypes may indiscriminately bind certain immune cells, increasing background noise. Finally, it is important to note whether a manufacturer has provided any validation data on a given antibody for its use in flow cytometry.
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The list of available fluorophores continues to grow, but it remains essential to consider key dye characteristics during panel design.
In this article, we explain why classic dyes retain their appeal and comment on the advantages modern fluorophores bring to flow cytometry-based research.