PolyQ Aggregation Assay to Identify Neuroprotective Effect of a Compound in Worms

Abstract

Source: Wang, Q. et al. Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity. J. Vis. Exp. (2021)

This video describes an in vivo method to screen the protective potential of neuroprotective test compounds in reducing polyQ aggregation in body wall muscle cells of nematodes and ameliorating aggregation-associated proteotoxicity.

Protocol

1. PolyQ aggregation assay

1. Preparation of nematodes for the polyQ aggregation assay
   1. Transfer 300-500 synchronized L1 larvae of AM141 to each well of a 48-well plate with 500 µL of S medium containing OP50 (OD₅₇₀ of 0.7-0.8) and 5 mg/mL of astragalan, typically one well per treatment for one time point.
   2. Seal the plate with parafilm and incubate at 20 °C and 120 rpm for 24, 48, 72, and 96 h.
   3. Harvest the nematodes in a sterile 1.5 mL microcentrifuge tube and wash with M9 buffer >3 times by centrifugation (1000 × g for 1 min) to remove the remaining OP50. Resuspend the AM141 nematodes in M9 b....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C. elegans strain
AM141 rmIs133 [unc-54p::Q40::YFP]	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/AM141
E. coli strain
OP50	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/OP50
Reagent
Sodium azide	Sinopharm Chemical Reagent Co., Ltd.	80115560	https://www.reagent.com.cn/goodsDetail/5e981aa807664e26af 551e96ff5f07cd
Equipment
384-well cell culture plate	Nest Biotechnology Co., Ltd.	761001	https://www.cell-nest.com/page94? _l=en&product_id=85
48-well cell culture plate	Nest Biotechnology Co., Ltd.	748001	https://www.cell-nest.com/page94? _l=en&product_id=85
Fluorescence microscope	Guangzhou Micro-shot Optical Technology Co., Ltd.	Mshot MF31-LED	https://www.mshot.com/ article/442.html
High-content imaging system	Molecular Devices	ImageXpress	https://www.moleculardevices.com/ products/cellular-imagingsystems#High-Content-Imaging
Microcentrifuge	GeneCompany	GENESPEED X1	https://www.genecompany.com/ index.php/Home/Goods/ goodsdetails/gid/189.html
Parafilm M	Sigma-Aldrich	P7793-1EA	https://www.sigmaaldrich.cn/CN/en/product/sigma/p7793?context=product
Microscope digital camera	Guangzhou Micro-shot Optical Technology Co., Ltd.	MS60	https://www.mshot.com/article/677.html
Software
Image acquisition and analysis software	Molecular Devices	MetaXpress	https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metaxpress