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Surface Barcoding of Live PBMC for Multiplexed Mass Cytometry.

Axel Ronald Schulz1, Henrik E Mei2

  • 1Mass Cytometry Lab, German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|May 12, 2019
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Summary

This study presents an optimized protocol for cell-surface barcoding of peripheral blood mononuclear cells (PBMCs) using palladium-labeled antibodies for mass cytometry. This method harmonizes data and enables precise immune phenotyping without interfering with standard assay channels.

Keywords:
Antibody-based sample barcodingCell-surface sample barcodingCyTOFImmune monitoringMass cytometryPalladiumβ2-Microglobulin

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Area of Science:

  • Immunology
  • Analytical Chemistry
  • Biotechnology

Background:

  • Mass cytometry is a powerful tool for high-dimensional single-cell analysis.
  • Harmonizing data from multiple samples in mass cytometry can be challenging.
  • Cell-surface barcoding offers a solution for pooling and processing multiple samples simultaneously.

Purpose of the Study:

  • To provide an optimized and validated protocol for cell-surface barcoding of peripheral blood mononuclear cells (PBMCs).
  • To enable the pooling of ten PBMC samples using a 5-choose-2 barcoding scheme with palladium-labeled antibodies.
  • To facilitate immune phenotyping by mass cytometry without compromising live cell surface marker analysis.

Main Methods:

  • Utilized palladium-labeled β2-microglobulin (B2M) antibodies for cell-surface barcoding.
  • Implemented a 5-choose-2 barcoding scheme for ten distinct PBMC samples.
  • Developed a method for generating palladium-labeled antibodies using amine-reactive isothiocyanobenzyl-EDTA (ITCB-EDTA).

Main Results:

  • Successfully barcoded ten PBMC samples using the described cell-surface antibody method.
  • Demonstrated that the palladium-based barcoding does not interfere with lanthanide channels used in mass cytometry.
  • Validated the protocol for immune phenotyping of live cells.

Conclusions:

  • The presented protocol offers an effective strategy for harmonizing mass cytometry data through cell-surface barcoding.
  • This method allows for multiplexed analysis of multiple samples, improving experimental efficiency.
  • The use of palladium-labeled antibodies provides a robust approach for antibody-based barcoding in mass cytometry.