Extended live-cell barcoding approach for multiplexed mass cytometry

Muharrem Muftuoglu,Junxiang Fang,Shaoheng Liang,Duncan Mak,Michael Andreeff,Li Li,Angelique J Lin,Jared K Burks,Ken Chen

doi:10.1038/s41598-021-91816-w

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.

Highlights

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition
ITCBE16 and mDOTA5 monomers are capable of chelating bivalent Pd isotopes, and monomers loaded with Pd isotopes can be successfully tagged to CD45, though it is more labor-intensive compared to generation of lanthanide-tagged antibodies using readily available mDTPA polymers
Both the Cd and Pd isotopes were loaded onto the MCP9 polymer after we demonstrated MCP9 polymer could capture bivalent Pd isotopes as well, and we used a 10-choose-2 combinatorial scheme

Summary

Introduction

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. 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. Both the Cd and Pd isotopes were loaded onto the MCP9 polymer after we demonstrated MCP9 polymer could capture bivalent Pd isotopes as well, and we used a 10-choose-2 combinatorial scheme This live-cell barcoding platform considerably expanded our ability to pool and barcode higher numbers of samples, which we describe

Objectives

Methods

Results

Conclusion