High-Throughput Multiplexed Single-Cell Analysis Using Protein Barcoding with Pacific Blue and DyLight800 2306498

Introduction High-throughput single-cell analysis requires strategies that minimize reagent use while enabling multiplexed detection of intracellular targets. Protein barcoding using spectrally distinct dyes offers a scalable approach to simultaneously analyze multiple samples under identical staining conditions. Here, we describe a workflow employing Pacific Blue and DyLight800 dyes for barcoding fixed and permeabilized cells, enabling multiplexed immunostaining and spectral unmixing on the CytoFLEX mosaic Spectral Detection module. Methods Cells were treated, fixed, and permeabilized prior to barcoding with 16 unique dye combinations of Pacific Blue and DyLight800. Barcoded samples were pooled and stained with antibodies targeting phosphorylated nuclear proteins conjugated to PE and APC. Enhanced green fluorescent protein (eGFP) transfection served as a reporter for transfection efficiency. Data acquisition was performed using the CytoFLEX mosaic Spectral Detection module, followed by computational unmixing and gating to resolve individual barcoded populations. Results Spectral profiles confirmed distinct intensity signatures for each barcoded combination, allowing accurate discrimination of up to 16 samples per well. Integration with plate-based workflows enabled processing of 1,536 samples per plate. Barcoding did not interfere with antibody binding or eGFP detection Conclusion Multiplexed analysis revealed consistent staining patterns across pooled samples, validating the approach for high-throughput intracellular signaling studies.