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

Abstract

Barcoded flow cytometry is a multiplexing technique allowing for the simultaneous acquisition of cells from different donors or experimental conditions in a highthroughput 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.

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells

“…Initial/prior live-cell barcoding of sample is therefore not only reducing technical variance but is also fully compatible with T-cell functional assays and can be used for pooled immunophenotyping. Anti-CD45 live-cell barcoding signal was stable over at least 16 h of culture most probably due to the absence of receptor internalization, similar to the observations of (Junker and Camillo Teixeira, 2022) and (Palchaudhuri et al, 2016).…”

“…Live-cell barcoding of fresh or thawed cells is compatible with both short-term and long-term T-cell activation assays in mouse ( Akkaya et al., 2016 ) and human cells, as it does not require fixation (our work) ( Junker and Camillo Teixeira, 2022 ). This is a commonly used strategy in immunological research to mark individual donors and thus assess pooled samples simultaneously in a high-throughput manner.…”

Hybrid Macromolecular Constructs as a Platform for Spectral Nanoprobes for Advanced Cellular Barcoding in Flow Cytometry

“…[7] In mass cytometry, the number of detection channels is (in principle) up to 130 but limited by the available metal tags [43] to 50, whereas conventional cytometry with 3-laser excitation uses 8-12 channels (clinical) [44] to 15 channels (research) and full spectrum flow cytometry extends the number of resolvable fluorochromes further to 18 parameters. [45] Barcoding six or more samples with distinct antibody-label conjugates means using up to six channels (systems of 5-choose-2 or 6-choose-3) [7,15] which is well tolerated in mass cytometry but impractical in conventional and full-spectrum flow cytometry. Moreover, those systems rely on the ultra-high expression of the barcoding target, since two or three reagents, compete for the same antigen epitope, and thus are limited for samples with a stable expression of the barcoding target, therefore excluding tumor samples.…”

“…Full spectrum cytometry can offer an alternative solution to overcome spreading error. [ 15 ] To reduce spreading error and preserve a reasonable number of possible barcodes, it is convenient to occupy a limited number of channels by using different intensities of barcodes. This is a convenient approach for fluorescent tags [ 4 ] but can be hardly achieved with live cell barcoding.…”

Association between NLRP3 inflammasome and periprocedural myocardial injury following elective PCI

“…Peripheral blood mononuclear cells (PBMCs) are taken as peripheral blood cells carrying a single round nucleus, including several classes of immune cells, such as lymphocytes, monocytes, dendritic cells and natural killer cells [ 10 , 11 ]. PBMCs are essential mediators of immune homeostasis and inflammation, and are widely used in immunological studies [ 12 ].…”