Barcoding of live peripheral blood mononuclear cells to assess immune cell phenotypes using full spectrum flow cytometry.

Barcoded flow cytometry is a multiplexing technique allowing for the simultaneous acquisition of cells from different donors or experimental conditions in a high-throughput manner. This approach allows to synchronize acquisition of samples and reduce variance introduced through the operator or technical platform. However, to date, only very few flow cytometry barcoding protocols have been developed, which often suffer from technical limitations. Here, we developed a novel barcoding protocol for a full-spectrum flow cytometry platform. We developed a 21-color immunophenotyping assay for up to 20 different samples analyzed simultaneously with comparable variance between repeated single-tube acquisition and postde-multiplexing. Barcoding offers great potential in parallelizing the analysis of complex cell populations such as peripheral blood mononuclear cells (PBMCs). Consequently, we assessed the performance of our method in situations where PBMCs were challenged with phytohaemagglutinin (PHA), a strong mitogen and broad activator of B cells and T cells, and superantigen Staphylococcus enterotoxin B (SEB) that has been reported to induce polyclonal T cell activation. PBMCs were either barcoded before pooled challenge or challenged individually pre-barcoding. Our final workflow included pooled immunophenotyping followed by machine learning aided single-cell data analysis and enabled us to identify robust PHA and SEB mode of action related phenotypic changes in PBMC immune cell lineages. Conclusively, we present a novel technique allowing the barcoded acquisition and analysis of PBMCs from up to 20 different donors and present a valid basis for the future development of complex immunophenotyping protocols.