Seeing the Confetti Colors in a New Light Utilizing Flow Cytometry and Imaging Flow Cytometry

Stochastic multicolor transgenic labeling systems, such as the Brainbow reporters, have emerged as powerful tools in lineage tracing experiments. Originally designed for large-scale mapping of neuronal projections in densely populated tissues, they have been repurposed for diverse uses. The Brainbow 2.1-derived Confetti reporter was used, for example, to define stem cell clonality and dynamics in crypts of the intestinal mucosa, T-cell clonality, microglial heterogeneity, and B-cell clonal evolution in germinal centers. Traditionally, read-outs have relied on imaging in situ, providing information about cellular localization within tissue stroma. However, recent applications of the technique have moved into hematopoietically derived motile cell types, for example, T and B lymphocytes and their progeny, creating an unmet need to survey larger populations of cells ex vivo to determine labeling densities or skews in color representation over time to read-out clonal expansion and selection effects. Originally designed for imaging methods, these reporters encode information in the spectral properties of fluorophores and their subcellular localization, making them poorly suited to traditional flow cytometry analyses. The advent of high-content imaging and imaging flow cytometry have recently closed the gap between flow cytometry and imaging. We analyzed a 10-color biallelic Confetti reporter using flow and imaging flow cytometry. Beyond its use as a high-throughput method for measuring reporter labeling densities and color distributions over time, it also opens the door to new avenues of research relying on similar read-outs, for example, tumor heterogeneity and clonal dynamics. © 2020 International Society for Advancement of Cytometry.