Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.
Methods Mol Biol
; 2696: 73-92, 2023.
Article
em En
| MEDLINE
| ID: mdl-37578716
ABSTRACT
Inflammasomes are crucial sentinels of the innate immune system that sense clues of infection, cellular stress, or metabolic imbalances. Upon activation, the inflammasome sensor (e.g., NLRP3) recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC rapidly oligomerizes to form a micron-sized structure termed "ASC speck." These are crucial for the activation of caspase-1 and downstream inflammatory signals released following a specific form of lytic cell death called pyroptosis. Hence, due to their considerably large size, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide three detailed protocols for imaging ASC specks (1) live-cell imaging of macrophage cell lines expressing a fluorescent protein fusion form of ASC, (2) imaging of human primary cells using immunofluorescence staining of endogenous ASC, and (3) visualization and quantification of specks on a single-cell level using imaging flow cytometry.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Adaptadoras de Sinalização CARD
/
Inflamassomos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article