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Conclusion:
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Here, we developed a barcoding protocol taking advantage of the increased resolution and minimized fluorochrome readout interference of full-spectrum flow cytometry. We present the development of a complex 21-colour immunophenotyping assay for up to 20 different samples analysed simultaneously. We show that the resolution of relatively rare T cell subsets is reproducible in samples from the same donor analysed conventionally or post-de-multiplexing. We did not detect multiplexing related batch effects when comparable appropriate fluorescence-minus controls were included in the protocol. The variance between repeated single-tube acquisition and post-de-multiplexing was comparable for lymphoid cell populations, and the method can be used for the simultaneous analysis of samples from the same or different donors. Finally, barcoding offers great potential in parallelising the analysis of complex samples where high inter-sample variability or rare populations are expected, such as immunophenotyping of peripheral blood mononuclear cells (PBMCs) challenged with an immunostimulant. Consequently, in order to assess activation induced effects on peripheral T cell phenotypes in a setting mimicking polyclonal, antigen-presenting cell induced T cell activation, we applied our assay protocol comparing superantigen Staphylococcus enterotoxin B (SEB) challenged cells from n=20 healthy donors in a multiplexed fashion. SEB mediated T cell activation is occasionally used for PD biomarker purposes char but so far has not extensively studied with regards to phenotypic immune cell changes at the population level. Our final workflow included barcoding and pooled immunophenotyping followed by machine learning aided single-cell data analysis to identify SEB driven changes in PBMC lineage composition with a special focus on T cell subsets. As a result, we identified 2 clusters of activated CD4+ T cells emerging exclusively upon superantigen challenge. These clusters would have been challenging to identify using conventional, non-multiplexed approaches as they were virtually absent in treatment-naïve cells, variable between donors and might hence be overlooked using manual gating strategies. Conclusively, we present a novel technique allowing the barcoded acquisition and analysis of PBMCs from up to 20 different donors and activation statuses and present a valid basis for the future development of complex immunophenotyping protocols.
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