Identification and isolation of splenic tissue resident macrophage subpopulations by flow cytometry

Abstract

Tissue resident macrophages in the spleen, including red pulp and white pulp macrophages, marginal zone macrophages (MZMs), and marginal zone metallophilic macrophages (MMMs), are highly heterogeneous as a consequence of adaptation to tissue-specific environments. Each macrophage subpopulation in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry. However, their phenotypical and functional characteristics remain incompletely understood due to the difficulty of identification and isolation by flow cytometry. We used a cocktail of three enzymes (Collagenase D, Dispase I and DNase I), rather than traditional mechanical grinding, for isolation of each subpopulation, which resulted in significant improvement of isolation of these macrophage subpopulations, particularly MZMs and MMMs, as determined by CD11b^hiF4/80^medTim4^hi and CD11b^hiF4/80^medTim4^med, respectively. This method should be helpful for molecular and functional characterization of each splenic resident macrophage subpopulation.