A Single 9-Colour Flow Cytometric Method to Characterise Major Leukocyte Populations in the Rat: Validation in a Model of LPS-Induced Pulmonary Inflammation

PLoS One. 2016 Jan 14;11(1):e0142520. doi: 10.1371/journal.pone.0142520. eCollection 2016.


The rat is a commonly used model for immunological investigation. Yet basic research and characterisation of leukocyte populations and sub-sets lags far behind murine research, with inconsistency on reported leukocyte markers and their overlap. These shortcomings limit the opportunity for more complex and advanced rat immunology research. In this study, we developed a robust 9-colour flow-cytometric protocol to elucidate the major blood and tissue rat leukocyte populations, and validated it in a model of LPS-induced pulmonary inflammation. Blood and tissues (lung, BALF, spleen, liver, bone marrow) from naïve Sprague-Dawley rats were collected and analysed by flow cytometry (FCM). Rats were exposed to aerosolised saline or LPS (1 mg/mL), at 3 and 24 hrs thereafter blood, lung and BALF were collected and analysed using FCM and ELISA. Neutrophils, two monocyte subsets, NK Cells, B Cells, CD4+, CD8+ T Cells and alveolar macrophages can be identified simultaneously across different tissues using a 9-colour panel. Neutrophils and monocytes can be distinguished based upon differential expression of CD43 and His48. Neutrophils and CD43Lo/His48Hi monocyte-macrophages are elevated in the lung at 3 and 24 hrs during LPS-induced pulmonary inflammation. This validated method for leukocyte enumeration will offer a platform for greater consistency in future rat immunology and inflammation research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bronchoalveolar Lavage Fluid / cytology
  • Bronchoalveolar Lavage Fluid / immunology
  • Cytokines / biosynthesis
  • Disease Models, Animal
  • Female
  • Flow Cytometry* / methods
  • Fluorescent Dyes
  • Immunophenotyping* / methods
  • Leukocytes / classification
  • Leukocytes / immunology
  • Leukocytes / metabolism*
  • Lipopolysaccharides / immunology
  • Pneumonia / immunology
  • Pneumonia / metabolism
  • Rats
  • Reproducibility of Results


  • Cytokines
  • Fluorescent Dyes
  • Lipopolysaccharides

Grants and funding

This work was funded by the Royal British Legion Centre for Blast injury Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.