Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments

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.