Measurement of rare innate immune cell activation directly in whole blood using imaging flow cytometry.

Abstract Signal transduction pathways regulate innate immune cell activation and thus have a critical influence on the effectiveness of host defense against pathogens. Binding to pathogen epitopes triggers signaling cascades that result in the translocation of transcription factors from the cytoplasm to the nucleus, which can be directly measured using microscopy. However, innate immune cells present significant challenges to image-based analysis due to their rarity and the need for simultaneous multispectral immunophenotyping. These challenges have historically required enrichment prior to imaging, even when using high speed imaging cytometry. Recent advancements in multispectral imaging flow cytometry have reduced these barriers, and in this study we show image-based measurement of NFkB activation in two innate immune cell types found in human whole blood. In the first set of experiments, we show dose and time kinetic activation of NFkB translocation specifically in monocytes by LPS and TNF-α. In the second set we show TLR-mediated activation of non-enriched circulating plasmacytoid dendritic cells, which represent less than 0.1% of periperal blood leukocytes. Our results in each of these systems uniquely demonstrate a method to perform image-base measurement of rare immune cell function directly in whole blood samples.