An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity 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)

In this video, we describe a method to evaluate the neuroprotective effect of a test compound, astragalan, on neuronal survival in Caenorhabditis elegans from polyQ-mediated neurotoxicity. The assay uses Caenorhabditis elegans expressing the abnormally expanded polyQ repeats fused to GFP in their ASH neurons to measure neuronal survival following test compound treatment.

Protocol

1. PolyQ-mediated neurotoxicity assay

1. Treatment of C. elegans with test samples
   1. To prepare nematodes for the polyQ neurotoxicity assay, transfer 300-500 synchronized L1 larvae of HA759 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 three replicate wells for each treatment.
   2. Seal the plate with parafilm and incubate at 15 °C and 120 rpm for 3 days.
   3. Collect the nematodes by centrifugation and wash 3-5 times with M9 buffer. Resuspend the nematodes in the M9 buffer for use in neuronal survival and avoidance assays.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<strong><em>C. elegans</em> strain</strong>
HA759 rtIs11 [osm-10p::GFP + osm-10p::HtnQ150 + dpy-20(+)]	Caenorhabditis&nbsp;Genetics Center (CGC)		https://cgc.umn.edu/strain/HA759
<strong><em>E. coli</em> strain</strong>
OP50	Caenorhabditis&nbsp;Genetics Center (CGC)		https://cgc.umn.edu/strain/OP50
<strong>Reagent</strong>
Agarose	Biowest	111860
Sodium azide	Sinopharm Chemical Reagent Co., Ltd.	80115560	https://www.reagent.com.cn/goodsDetail/5e981aa807664e26af<br/> 551e96ff5f07cd
<strong>Equipment</strong>
48-well cell culture plate	Nest Biotechnology Co., Ltd.	748001	https://www.cell-nest.com/page94?_l=en&amp;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 Pico	https://www.moleculardevices.com/products/cellular-imaging-systems#High-Content-Imaging
Microcentrifuge	GeneCompany	GENESPEED X1	https://www.genecompany.com/index.php/Home/Goods/goodsdetails/gid/189.html
Microscope digital camera	Guangzhou Micro-shot Optical Technology Co., Ltd.	MS60	https://www.mshot.com/article/677.html
Microwave	Midea Corp.	&nbsp;M1-211A	https://www.midea.cn/10000/10000000001
Parafilm M	Sigma-Aldrich	P7793-1EA	https://www.sigmaaldrich.cn/CN/en/product/sigma/p7793?context=product
<strong>Software</strong>
Image acquisition and analysis software	Molecular Devices	MetaXpress	https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metaxpress