Measuring NLR oligomerization II: Detection of ASC speck formation by confocal microscopy and immunofluorescence
Journal Title
Methods Mol Biol
Publication Type
Journal Article
Abstract
Inflammasome assembly results in the formation of a large intracellular protein scaffold driven by the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). Following inflammasome activation, ASC polymerizes to form a large singular structure termed the ASC "speck," which is crucial for recruitment of caspase-1 and its inflammatory activity. Hence, due to the considerably large size of these structures, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide two detailed protocols for imaging ASC specks: by (1) live-cell imaging of monocyte/macrophage cell lines expressing a fluorescently tagged version of ASC and (2) immunofluorescence of endogenous ASC in cell lines and human immune cells. In addition, we outline a protocol for increasing the specificity of ASC antibodies for use in immunofluorescence.
Publisher
Springer
Research Division(s)
Inflammation
PubMed ID
27221487
Terms of Use/Rights Notice
Refer to copyright notice on published article.


Creation Date: 2016-06-16 10:27:03
Last Modified: 2016-06-16 10:28:35
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