Extended live-cell barcoding approach for multiplexed mass cytometry

Abstract

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. Sample pooling after prior surface staining termed live-cell barcoding is more desirable than intracellular barcoding, where samples are pooled after fixation and permeabilization, since it does not depend on fixation-sensitive antigenic epitopes. In live-cell barcoding, the general approach uses two tags per sample out of a pool of antibodies paired with five palladium (Pd) isotopes in order to preserve appreciable signal-to-noise ratios and achieve higher yields after sample deconvolution. The number of samples that can be pooled in an experiment using live-cell barcoding is limited, due to weak signal intensities associated with Pd isotopes and the relatively low number of available tags. Here, we describe a novel barcoding technique utilizing 10 different tags, seven cadmium (Cd) tags and three Pd tags, with superior signal intensities that do not impinge on lanthanide detection, which enables enhanced pooling of samples with multiple experimental conditions and markedly enhances sample throughput.

Transgenic Expression of IL15 Retains CD123-Redirected T Cells in a Less Differentiated State Resulting in Improved Anti-AML Activity in Autologous AML PDX Models

“…Cells from murine bone marrow and spleens were barcoded and pooled before surface staining with a panel of 52 antibodies (metal isotope-conjugated, Table S1 ) and were analyzed on a Helios mass cytometer (Fluidigm) as reported previously ( 17 , 18 ). Using a cloud-based computational platform OMIQ.ai (Omiq, Inc. Santa Clara, CA), data were normalized and analyzed with gating on CD45 + cells.…”

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

“…A frequently used approach relies on barcoding individual (live or fixed) PBMC samples with a unique combination of anti-CD45 antibodies [16][17][18][19] or other constitutively expressed cell surface markers such as CD298 and beta-2-microglobulin [20]. Through combinatorial mathematics, many samples can thus be acquired at the same time [21]. For example, if seven anti-CD45 antibodies are available for barcoding, three of which are used to co-label PBMCs of one specific donor/condition, this allows for up to 35 unique barcoding combinations ("7-choose-3").…”

Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding

“…These results confirm that our hybrid-tag nanotrackers are as suitable barcoding reagents for heterogeneous cell populations such as blood cells—as other reported mass cytometry reagents. 6 , 11 , 17 , 22 , 32 , 33 However, a key differential aspect of our approach is that these hybrid-tag nanotrackers integrate both fluorescence and mass cytometry cell barcoding in a single device and allow a dual-modal readout. Based on these results, we can conclude that these hybrid-tag nanotrackers are suitable for barcoding of both heterogeneous and homogeneous cell populations.…”

“…This fact makes these reagents unsuitable for long-term culture assays. To overcome this issue, a large number of reagents were developed to barcode cell surface molecules such as CD45, the β-macroglobulin subunit of major histocompatibility complex (MHC) class 1, and the β-3 subunit of the sodium/potassium ATPase. − On the other hand, cell-compatible small probes have been investigated such as osmium and ruthenium in the form of oxides or maleimide-functionalized tellurophene probe, TeMal, − allowing cell barcoding not only in live cells but also in permeabilized cells. Notably, none of the barcoding reagents described above can be used in long-term drug response studies, whereby cells need to be prebarcoded just before a mass cytometry study begins. , Some efforts have been focused on the development of dual reagents, but none has been reported so far for flow and mass cytometry dual-modal readout in longitudinal cell assays and multiplexed drug studies. , …”