Why can't they play nicely? Navigating unexpected challenges in designing new flow cytometry panels and expanding existing ones

In recent decades, the field of cytometry has witnessed a remarkable surge in the adoption of new fluorescent dyes, driving the widespread integration of large panels and spectral technologies into daily workflows. These developments have underscored the critical need to account for dye interactions within the reagent mix and during cell staining procedures. However, these interactions are often not sufficiently described, primarily due to the scarcity of unbiased technical reports, further compounded by the proprietary nature of reagent information. In this tutorial, we will first briefly examine the chemistry families of fluorescent dyes commonly employed in flow cytometry. We will then revisit established considerations regarding unwanted interactions between reagents, as well as between dyes and cells. This includes discussions on Fc receptor binding, cyanine- and cyanine-like dye interactions with myeloid cells, polymer dye aggregation, undesired FRET detection, and other pertinent factors. The primary emphasis of the tutorial will center on what the community regards as 'novel dyes' and their potential unwanted interactions with both reagent mix components and target cells. Furthermore, we will explore strategies for future-proofing panel designs to accommodate the addition of reagents, as well as addressing adding entities with varying brightness levels to the panels. These discussions will provide attendees with valuable insights into optimizing flow cytometry panel designs to effectively navigate the evolving landscape of cytometric analyses.

CMLE Credit: 1.0