Ratiometric Barcoding for Mass Cytometry

Abstract

Barcoding is of importance for high-throughput cellular and molecular analysis. A ratiometric barcoding strategy using lanthanide-coordinated polymer dots (Ln-Pdots) was developed for mass cytometric analysis. By using 3 metal isotopes and 4 ratio intensity levels, 16 barcodes were generated to code, and later decode, cell samples in mass cytometry. The ratiometric Ln-Pdot barcodes not only provided high-mass-signal intensities but also eliminated the bias caused by different concentrations of the labeling reagents/barcodes and run-to-run differences in cell labeling efficiency. The ability to distinguish clearly the 16 sets of labeled MCF-7 cells with mass cytometry demonstrated the excellent resolving power of the ratiometric Ln-Pdot barcodes. Furthermore, the results from barcoding PBMC samples via CD45-specific cellular targeting indicated that the ratiometric Ln-Pdot barcodes could facilitate mass cytometry in high-throughput and multiplexed analysis, especially with precious human samples.

Figure 1.

(A) Histogram showing the size distribution of Pdots-(5,5), determined by analyzing >100 particles in TEM images (B). The mean diameter was 31.2 ± 6.8 nm. (B) TEM images of Pdots-(5,5). The absorption (C) and fluorescence (D) spectra of Pdots-(5,5). (E) Cell viability of MCF-7 cells after incubation with different concentrations of barcoding Ln-Pdots for 24 hours. (F) The barcodes generated by Ln-Pdots with three lanthanide isotopes and four different intensity ratios; the isotopic intensity ratios were measured with ICP-MS.

Figure 2.

Distribution of 16 sets of MCF-7 cells labeled with different Ln-Pdots barcodes via endocytosis and then analyzed by CyTOF. (A-C) Mass cytometry measurements of the mass intensity distributions of MCF-7 cells labeled with Pdot-(5, 5): (A) Tb¹⁵⁹, (B) Ho¹⁶⁵, and (C) Tm¹⁶⁹ channels. (D) Discrimination of 16 sets of MCF-7 cells individually using mass cytometry after coding with 16 Pdot barcodes as described in Figure 1F. The ratios of Tb/Tm and Ho/Tm were plotted for each cell set. (E) Mass cytometric analysis of the mixed samples coded with Pdot-(1, 5), Pdot-(5, 5), Pdot-(10, 5), Pdot-(5, 1), and Pdot-(5, 10). (F) Mass cytometric analysis of the mixed samples coded with Pdot-(0.1, 10), Pdot-(0.1, 5), Pdot-(0.1, 1), Pdot-(0.1, 0.1), Pdot-(1, 0.1), Pdot-(5, 0.1), and Pdot-(10, 0.1).

Figure 3.

(A) Schematic diagram depicting the barcoding of different samples and labeled with different commercially available mass tags. (B) De-barcoding by mass cytometric analysis the mixed samples in (A).

Scheme 1.

Schematic showing the ratiometric barcodes based on Ln-Pdots for multiplex mass cytometric analysis. The Ln-Pdot barcodes can label cells either through endocytosis or specific antigen-antibody binding.