Abstract
The cell envelopes of pathogens comprise a wealth of unique glycolipids, which are important modulators of the host immune responses during infection and in some cases have been used as adjuvants. Despite this abundant basic knowledge, the identities of the host immune receptors for mycobacterial lipids have long been elusive (Ishikawa et al., Trends Immunol 38:66-76, 2017). We describe the method of how to isolate glycolipids from microorganisms and how to analyze the glycolipids' potential to activate reporter cells and bone marrow-derived dendritic cells (BMDCs), such as surface marker expression and reactive oxygen species (ROS) production. Additionally, we outline an in vitro BMDC/T cell coculture model to investigate functional consequences of leukocyte activation, such as cytokine production. In this chapter, we provide a guide for extracting glycolipids from microorganisms and how to use them to activate leukocytes. We also present methods on how to generate and activate reporter cells, as well as BMDCs and how to set up BMDC/T cell cocultures. We further outline how to generate samples and how to analyze the immunomodulatory effect glycolipid exposure has on these cells, via flow cytometry, ROS production assays and ELISA.
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