Abstract
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 (1mg/mL), at 3 and 24hrs 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 24hrs 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.
Highlights
Reproducible and robust characterisation of leukocytes is necessary for advanced immunological and inflammation research
We utilised the FSC-H and FSC-W parameters to identify and exclude cells stuck together and larger clumps. This resulted in a population of live single leukocytes which were sequentially separated, first for T lymphocytes by CD3 (v) which were further delineated by CD4 and CD8 (vi)
The same gating strategy was applied successfully to lung tissue (Fig 1B i-x), we were able to identify Alveolar Macrophages based on their unique pattern of autofluorescence
Summary
Reproducible and robust characterisation of leukocytes is necessary for advanced immunological and inflammation research. Studies conducted in rat are currently limited by the lack of an accurate and validated method that permits simultaneous quantification of major leukocyte subsets. Neutrophils are the primary first responder to inflammation [1]. Following release from the bone marrow these cells are capable of circulating in the periphery [2], marginating in the PLOS ONE | DOI:10.1371/journal.pone.0142520. Major Rat Leukocyte Populations: LPS Validation analysis, decision to publish, or preparation of the manuscript Following release from the bone marrow these cells are capable of circulating in the periphery [2], marginating in the PLOS ONE | DOI:10.1371/journal.pone.0142520 January 14, 2016
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