Measuring NLR oligomerization II: Detection of ASC speck formation by confocal microscopy and immunofluorescence
- Author(s)
- Beilharz, M; De Nardo, D; Latz, E; Franklin, BS;
- 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
- Publisher's Version
- https://doi.org/10.1007/978-1-4939-3566-6_9
- 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