Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding

[Image: see text] Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.