Multi-parametric quantification of monocytes using live-cell analysis

Abstract

Abstract Monocytes play critical roles in innate immunity by migrating to inflamed tissue, where they clear micro-organisms and apoptotic cells, repair injured tissues, and recruit other immune cells. These highly plastic cells can change their functional phenotypes in response to a variety of cellular signals, including the Akt signaling pathway. Here we provide robust in vitro assays for the kinetic evaluation of monocytes and exemplify how these further our understanding of their biological roles. The Incucyte ®Live-Cell Analysis System was used to acquire phase and fluorescence images of monocytic cell lines, which were automatically analyzed using integrated software. To assess immune cell activation, RAW264.7 monocytes were treated with lipopolysaccharide (LPS) and phenotypic morphological changes were quantified using label-free analysis. These data showed a concentration-dependent increase in activated cells as they become larger and flatter, with a maximal response of 56% for 100 ng/mL LPS at 33 hours. To monitor Akt activity, we used cells expressing the Incucyte ®Kinase Akt Green/Red biosensor, a genetically encoded fluorescent reporter whose subcellular localization is dependent on Akt activation. Cytokine analysis was performed using the iQue ®Advanced Flow Cytometer and a custom Qbeads ®PlexScreen Kit. LPS induced a rapid concentration-dependent activation of Akt (EC 501.31 ng/mL) and stimulated release of inflammatory proteins TNFα and CCL3 (MIP-1α), consistent with LPS-inducing activation via the release of pro-inflammatory agents. These data exemplify that live-cell analysis, alongside advanced analytical methods, is a powerful approach enabling multi-parametric quantification of immune cells.