Abstract
Internalization of cell surface Toll-like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.
Keywords: TLR4, LPS, TRIF, CD14, Endocytosis, Peritoneal macrophages
Background
Toll-like Receptor 4 (TLR4) is unique among the Toll-like receptors in that it can deliver qualitatively different signals from two distinct cellular locations (reviewed in Takeda and Akira, 2004). Cell surface TLR4/MD2 is initially ligated by extracellular bacterial lipopolysaccharide (LPS) and engages the intracellular adaptor MAL and MyD88. Subsequently a fraction of cell surface LPS bound TLR4/MD2 will undergo CD14 and clathrin dependent internalization and translocation to an early endosomal compartment (Zanoni et al., 2011). In this endosomal compartment TLR4, engages the signaling adaptors TRAM and TRIF. Much of the regulation of TLR4 internalization and transport remains to be discovered. A flow cytometric method for quantifying loss of TLR4 surface expression, as a metric for endosomal translocation was first described in Bone Marrow Derived Macrophages (BMDMs) by Jonathan Kagan and colleagues (Kagan et al., 2008). Here we describe a modified version of this protocol to enable quantitation of TLR4 internalization in thioglycollate-elicited peritoneal macrophages on which we have published (Rajaiah et al., 2015; Perkins et al., 2018).
Materials and Reagents
Equipment
Software
Procedure
Day 1
Day 2
Data analysis
Notes
Recipes
Acknowledgments
Supported by the US National Institutes of Health (AI123371 and AI125215 to S.N.V.). This protocol was adapted or modified from previous work (Kagan et al., 2008; Zanoni et al., 2011; Rajaiah et al., 2015).
Competing interests
We declare there are no competing interests related to this work.
References
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