Related Experiment Video
Updated: Mar 22, 2026
High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence
Published on: October 28, 2025
Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments
Adeeb H Rahman1,2,3, Leticia Tordesillas3,4, M Cecilia Berin3,4
1Human Immune Monitoring CoRE, Icahn School of Medicine at Mount Sinai, New York, New York, 10029.
Insights
Non-specific antibody binding in eosinophils during intracellular mass cytometry (CyTOF) can be blocked using heparin. This simple protocol ensures accurate analysis of intracellular proteins in eosinophils.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Mass cytometry (CyTOF) is a powerful tool for analyzing heterogeneous cell populations.
- Accurate CyTOF analysis depends on specific antibody-antigen binding.
- Intracellular CyTOF of human whole blood samples can be affected by experimental artifacts.
Purpose of the Study:
- To identify and address a previously unappreciated artifact in intracellular CyTOF analysis.
- To investigate non-specific antibody binding in human eosinophils during CyTOF.
- To develop a protocol to mitigate this artifact for accurate intracellular protein assessment.
Main Methods:
- Intracellular mass cytometry (CyTOF) analysis of human whole blood samples.
- Investigated non-specific antibody binding to eosinophils.
- Developed and tested a blocking protocol using heparin to reduce non-specific staining.
Main Results:
- Identified non-specific antibody binding to eosinophils during intracellular CyTOF.
- Hypothesized and confirmed a charge-based interaction between metal-labeled antibodies and cationic eosinophil granule proteins.
- Demonstrated that heparin, an anionic protein, effectively blocks this non-specific binding to background levels.
Conclusions:
- A simple heparin blocking protocol significantly reduces non-specific antibody staining in eosinophils during intracellular CyTOF.
- This method eliminates a source of error, enabling accurate assessment of intracellular proteins in eosinophils.
- The protocol is applicable to all experiments involving intracellular staining of human whole blood samples by CyTOF.
Abstract:
The analysis of heterogeneous cell samples by mass cytometry (CyTOF) relies on the assumption that metal labeled antibodies accurately bind to their target antigens. We report a previously unappreciated experimental artifact of non-specific antibody binding by eosinophils during intracellular CyTOF analysis of human whole blood samples. We hypothesized that this non-specific binding results from a charge-based interaction between the metal-labeled antibodies and highly cationic proteins found in eosinophillic granules and found that this non-specific staining artifact could be reduced to background levels with a simple blocking protocol using heparin as a competing anionic protein. This protocol eliminates a potential source of erroneous data interpretation in all experiments involving intracellular staining of human whole blood samples, and allows accurate assessment of dynamic changes in intracellular proteins in eosinophils by CyTOF. © 2016 International Society for Advancement of Cytometry.
