Protocol to measure apoptosis-associated speck-like protein containing a CARD specks in human cerebrospinal fluid via imaging flow cytometry

• Published in: STAR protocols 2024-03, Vol.5 (1), p.102916-102916, Article 102916
• Main Authors: Sánchez, Kathryn E., Jiang, Shanya, Palencia Desai, Sharina, Thompson, Jeffery, Hobson, Sasha, Rosenberg, Gary A., Bhaskar, Kiran
• Format: Article
• Language: English
• Subjects: Amyloid beta-Peptides - metabolism; Apoptosis; CARD Signaling Adaptor Proteins - metabolism; Clinical Protocol; Flow Cytometry; Flow Cytometry - methods; Humans; Inflammasomes - metabolism; Molecular Biology; Neuroscience; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.102916

Apoptosis-associated speck-like protein containing a c-terminal caspase activation and recruitment domain (ASC) specks are elevated in the cerebrospinal fluid (CSF) of Alzheimer’s disease and related dementias (AD/ADRDs) patients. Here, we present a flow cytometry protocol to quantify ASC specks. We describe steps for fluorescently labeling ASC specks using antibody technology, visualizing with imaging flow cytometry, and gating based on physical characteristics. CSF ASC specks levels positively correlate with phosphorylated tau (Thr181) and negatively correlate with amyloid β ratio (42/40), thus serving as a neuroinflammatory biomarker for diagnosing AD/ADRDs. For complete details on the use and execution of this protocol, please refer to Jiang et al.1 [Display omitted] •Purification of ASC specks from ASC-cerulean inflammasome reporter macrophages•Quantification of ASC specks in human cerebrospinal fluid with flow cytometry•Purified ASC specks and ASC knockout tissue are used to validate the methodology•ASC specks correlate with phosphorylated tau (T181) and amyloid β ratio in the CSF Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Apoptosis-associated speck-like protein containing a c-terminal caspase activation and recruitment domain (ASC) specks are elevated in the cerebrospinal fluid (CSF) of Alzheimer’s disease and related dementias (AD/ADRDs) patients. Here, we present a flow cytometry protocol to quantify ASC specks. We describe steps for fluorescently labeling ASC specks using antibody technology, visualizing with imaging flow cytometry, and gating based on physical characteristics. CSF ASC specks levels positively correlate with phosphorylated tau (Thr181) and negatively correlate with amyloid β ratio (42/40), thus serving as a neuroinflammatory biomarker for diagnosing AD/ADRDs.