Abstract
Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.
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Acknowledgements
We thank G. Jager for administrative support and A. Jager for mass cytometry quality control and instrument maintenance. We thank J. Ramunas, S.-Y. Chen, V.D. Gonzalez, and E.R. Zunder for valuable discussions. This work was supported by grants from the US National Institutes of Health (U19 AI057229, 1U19AI100627, R01CA184968, R33 CA183654, and R33 CA183692), US National Heart, Lung, and Blood Institute (N01-HV-00242), US Department of Defense (OC110674 and 11491122), Bill & Melinda Gates Foundation (OPP1113682), and Food and Drug Administration (HHSF223201210194C).
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G.H. developed EMTs and conjugation protocols, performed the experiments, and wrote the manuscript. M.H.S. performed mouse experiments with bismuth mass tags and performed data analysis. S.C.B. provided advice and assistance with the manuscript. W.J.F. provided advice and assistance with the manuscript. G.P.N. helped design the experiments and edited the manuscript.
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G.P.N. declares that he had a personal financial interest in Fluidigm, the manufacturer of the mass cytometer used in this study, for the duration of this project. The remaining authors declare no competing interests.
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1. Han, G. et al. Cytometry A 91, 1150–1163 (2017): https://doi.org/10.1002/cyto.a.23283
2. Porpiglia, E. et al. Nat. Cell Biol. 19, 558–567 (2017): https://doi.org/10.1038/ncb3507
Integrated supplementary information
Supplementary Figure 1 Pearson correlations between bismuth- and lanthanide-tagged antibodies.
a) Linear regression of marker intensity using 209Bi- and170Er-tagged CD3 antibodies. Log10 mean value of seven marker intensities in Jurkat cells. Pearson correlation, r = 0.997; P < 0.00001, two-tailed t test. (b) Linear regression of marker intensity using 209Bi- and 176Yb-tagged CD56 antibodies. Log2 mean value of 40 marker intensities in 15 cell subsets from human PBMCs normalized to minimal marker value in each cell subset. The data includes 600 combinations, 15 populations x 40 markers. Pearson correlation, r = 0.991; P < 0.00001, two-tailed t test.
Supplementary Figure 2 Manual gating strategy for PBMCs using biaxial scatter plots.
The gating hierarchy is demonstrated for 15 manually gated cell populations, which are used in the viSNE plots and heat maps in Figure 9. All gates were used with Boolean “AND” logic in Cytobank software.
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Supplementary Figures 1 and 2
Supplementary Table 1
The panel of antibodies, reporter isotopes, and concentrations in CyTOF assays
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Han, G., Spitzer, M.H., Bendall, S.C. et al. Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry. Nat Protoc 13, 2121–2148 (2018). https://doi.org/10.1038/s41596-018-0016-7
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DOI: https://doi.org/10.1038/s41596-018-0016-7
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