Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments

Cytometry A. 2016 Jun;89(6):601-7. doi: 10.1002/cyto.a.22826. Epub 2016 Apr 6.

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.

Keywords: CyTOF; eosinophil; granulocyte; heparin; mass cytometry; nonspecific antibody binding; whole blood.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies / chemistry*
  • Antigens / chemistry
  • Antigens / immunology
  • Artifacts
  • Binding, Competitive
  • Child
  • Cytoplasmic Granules / chemistry
  • Cytoplasmic Granules / immunology
  • Eosinophil Cationic Protein / antagonists & inhibitors
  • Eosinophil Cationic Protein / chemistry*
  • Eosinophils / chemistry
  • Eosinophils / cytology*
  • Eosinophils / immunology
  • Flow Cytometry / instrumentation
  • Flow Cytometry / standards*
  • Heparin / chemistry*
  • Humans
  • Mass Spectrometry / instrumentation
  • Mass Spectrometry / standards*
  • Primary Cell Culture
  • Protein Binding
  • Staining and Labeling / methods
  • Staining and Labeling / standards*

Substances

  • Antibodies
  • Antigens
  • Heparin
  • Eosinophil Cationic Protein