title: Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay.
abstract: The NLRP3 inflammasome is assembled in macrophages and monocytes in response to inflammatory and danger stimuli. The atypical nature of the NLRP3 complex impedes detection of NLRP3 inflammasome formation by conventional biochemical and cell biology methods. In situ proximity ligation assay (PLA) provides an alternative method of detection, localization, and quantification of protein-protein interactions in tissue and cell samples. Two primary antibodies raised in different species detect the two proteins of interest. When the proteins are in close proximity, secondary antibodies conjugated with specific DNA probes hybridize with linking oligonucleotides to form a DNA bridge between the two proteins. Amplification of the DNA bridge then facilitates detection by microscopy using fluorescence probes. Here, we describe application of in situ PLA to detect NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages and human monocyte cell line THP1.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan.
authors.forename: Yung-Hsuan
authors.initials: YH
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan. mblai@gate.sinica.edu.tw.|Institute of Immunology, National Taiwan University, Taipei, Taiwan. mblai@gate.sinica.edu.tw.
authors.forename: Ming-Zong
authors.initials: MZ
authors.lastname: Lai
mesh_terms: D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D000074084:THP-1 Cells
chemical_list: D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; C492716:Nlrp3 protein, mouse; D004247:DNA
keywords: IL-1β; In situ proximity ligation assay; Inflammasome; Macrophages; NLRP3 complex; NLRP3-ASC-caspase-1; Protein–protein interaction