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Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells
Published on: June 19, 2019
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Clickable and High-Sensitivity Metal-Containing Tags for Mass Cytometry
Bedilu Allo1, Xudong Lou1, Alexandre Bouzekri1
1Fluidigm Canada Inc. , Markham , Ontario L3R 4G5 , Canada.
Bioconjugate Chemistry
|May 8, 2018
Summary
A new click chemistry method enables metal tagging of diverse biomolecules for mass cytometry, overcoming limitations of previous thiol-maleimide chemistry. This advance improves sensitivity and specificity for applications in proteomics and glycomics.
Area of Science:
- Bioconjugation Chemistry
- Mass Cytometry Reagent Development
- Single-Cell Analysis Technologies
Background:
- Mass cytometry relies on metal-tagged reagents for multiplexed single-cell biomarker identification.
- Current thiol-maleimide chemistry for antibody conjugation requires antibody reduction and is limited for certain antibody classes (e.g., IgM) and cysteine-lacking biomolecules.
- This limitation hinders the broad application of mass cytometry in various biological fields.
Purpose of the Study:
- To develop a novel conjugation strategy for metal tagging a wider range of biomolecules and affinity reagents.
- To overcome the limitations of existing thiol-maleimide chemistry in mass cytometry reagent preparation.
- To enhance sensitivity and specificity of metal-tagged reagents for advanced single-cell analysis.
Main Methods:
- Developed a new metal-chelating polymer (MCP) conjugation approach using click chemistry (copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene cycloaddition).
- Activated biomolecules with amine groups using dibenzocyclooctyne or tetrazine moieties for conjugation with azide- or transcyclooctene-terminated MCPs.
- Generated dual-tagged reagents for multi-modal detection (immunofluorescence, mass cytometry, imaging mass cytometry) and tested on Helios and Hyperion Imaging Systems.
Main Results:
- Successfully generated highly sensitive and specific metal-tagged IgGs, IgMs, peptides, and lectins.
- Demonstrated the utility of click-conjugated reagents for immunophenotyping, glycobiology, and tissue imaging.
- Observed superior sensitivity and specificity in some click-conjugated reagents compared to thiol-maleimide derived ones.
Conclusions:
- The novel click chemistry-based conjugation strategy effectively labels a diverse array of biomolecules for mass cytometry.
- This approach overcomes previous limitations, enabling broader applications in proteomics and glycomics.
- The developed metal-tagged reagents offer enhanced sensitivity and specificity for advanced single-cell and tissue analysis.
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