Development and Validation of a Flow Cytometric Method to Evaluate Phagocytosis of pHrodo™ BioParticles® by Granulocytes in Multiple Species

J Immunol Methods. 2013 Apr 30;390(1-2):9-17. doi: 10.1016/j.jim.2011.06.027. Epub 2011 Jul 13.

Abstract

Phagocytosis plays a pivotal and essential role in host immune defense, both as a focal constituent of the innate immune response and a bridging element linking innate and adaptive immunity. Phagocytosis has been demonstrated to be critical in development, tissue remodeling, wound healing and resolution of inflammation through clearance of foreign organisms, apoptotic cells and the production of anti-inflammatory mediators. During pre-clinical investigations, therapeutic drug candidates may alter host resistance to infectious agents by modulating the immune system. The assessment of phagocytic function can be a critical parameter of immunotoxicology for this adverse effect. Utilizing pH-sensitive pHrodo™ BioParticles®, a flow cytometric phagocytosis method was developed and validated in rodent and non-human primate (NHP) species under rigorous GLP compliant procedures. Using species-specific granulocyte markers as well as appropriate temperature and pharmacologic controls, we have developed an ex vivo assay to measure phagocytic function. The method has been optimized to utilize minimal sample volume of whole blood. The assay represents a rapid and reliable tool that can be implemented to evaluate the immunotoxic and immunomodulatory effects of therapeutic candidates.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Cytochalasin D / pharmacology
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / immunology
  • Female
  • Flow Cytometry / methods*
  • Granulocytes / immunology*
  • Granulocytes / metabolism
  • Macaca fascicularis
  • Macrolides / pharmacology
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Phagocytosis / drug effects
  • Phagocytosis / immunology*
  • Reproducibility of Results

Substances

  • Enzyme Inhibitors
  • Macrolides
  • Nucleic Acid Synthesis Inhibitors
  • Cytochalasin D
  • bafilomycin A1