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

Samenvatting

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.
2. Preparation of agarose pad
   1. Add 2 g of agarose to 100 mL of M9 buffer (2%, w/v) and heat the agarose solution in a microwave to near-boiling.
   2. Dispense 0.5 mL of melted agarose onto the center of a 1 mm thick microscopy glass slide placed between two pieces of 2 mm thick glass plates. Cover with another slide vertically. Once the agarose cools down and is solidified, gently remove the top slide.
3. ASH neuronal survival assay
   1. Add a drop of 20 mM sodium azide onto the agarose pad. Transfer 15-20 HA759 nematodes into the drop to immobilize them.
   2. Place a coverslip gently over the nematodes. Keep the slide under a fluorescence microscope fitted with a digital camera. Select a 40x objective lens and FITC filter to detect GFP-positive ASH neurons in the head region of the nematodes.
   3. Select more than 50 nematodes in each group randomly to count the number of nematodes with GFP-labeled bilateral ASH neurons in their head region. Calculate the survival rate of ASH neurons:
      Neuronal survival (%) =
      Where N_survival and N_total are the number of nematodes with GFP-positive ASH neurons and the total number of tested nematodes in each group, respectively.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
<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