High-dimensional multi-pass flow cytometry via spectrally encoded cellular barcoding

Advances in immunology, immuno-oncology, drug discovery and vaccine development demand improvements in the capabilities of flow cytometry to allow it to measure more protein markers per cell at multiple timepoints. However, the size of panels of fluorophore markers is limited by overlaps in fluorescence-emission spectra, and flow cytometers typically perform cell measurements at one timepoint. Here we describe multi-pass high-dimensional flow cytometry, a method leveraging cellular barcoding via microparticles emitting near-infrared laser light to track and repeatedly measure each cell using more markers and fewer colours. By using live human peripheral blood mononuclear cells, we show that the method enables the time-resolved characterization of the same cells before and after stimulation, their analysis via a 10-marker panel with minimal compensation for spectral spillover and their deep immunophenotyping via a 32-marker panel, where the same cells are analysed in 3 back-to-back cycles with 10–13 markers per cycle, reducing overall spillover and simplifying marker-panel design. Cellular barcoding in flow cytometry extends the utility of the technique for high-dimensional multi-pass single-cell analyses.