Phagocyte interactions with Mycobacterium tuberculosis — Simultaneous analysis of phagocytosis, phagosome maturation and intracellular replication by imaging flow cytometry

•We present a method for studying M. tuberculosis-infected macrophages.•The method is based on imaging flow cytometry and is quantitative and rapid.•Phagocytosis, phagosome maturation and bacterial replication can be analyzed.•The main advantage is that several factors are analyzed simultaneously.•The method has basic research and screening applications.

Utilization of compounds that enhance the innate immune response against Mycobacterium tuberculosis is an attractive strategy for combating tuberculosis in the post-antibiotic era. Thus, it is crucial to develop methods that can be used to screen for such compounds and to investigate their mechanisms of action. Here, we used imaging flow cytometry (ImageStreamX Mk II), which enables rapid quantification of microscopic images in flow, to study the interaction between phagocytes and M. tuberculosis. Macrophage-differentiated THP-1 cells were infected with GFP-expressing M. tuberculosis H37Ra, and methods for rapidly assessing phagocytosis, phagosome maturation, and bacterial replication inside the cells were developed and evaluated. These aspects of innate immunity are essential in determining the outcome of mycobacterial infection of phagocytes. The technique was found effective for monitoring phagocytosis of mycobacteria, phagosomal acidification and phagolysosomal fusion, as well as for measuring mycobacterial replication inside the cells. Several of these aspects could be analyzed simultaneously in the same sample, providing a great deal of information about the phagocyte–mycobacterial interaction at once. Thus, this method has great potential to be useful both for basic research questions and for evaluating compounds that enhance the innate immune response against M. tuberculosis.