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

Protocolo

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.

Materiais

Name	Company	Catalog Number	Comments
C. elegans strain
HA759 rtIs11 [osm-10p::GFP + osm-10p::HtnQ150 + dpy-20(+)]	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/HA759
E. coli strain
OP50	Caenorhabditis Genetics Center (CGC)		https://cgc.umn.edu/strain/OP50
Reagent
Agarose	Biowest	111860
Sodium azide	Sinopharm Chemical Reagent Co., Ltd.	80115560	https://www.reagent.com.cn/goodsDetail/5e981aa807664e26af 551e96ff5f07cd
Equipment
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 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.	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
Software
Image acquisition and analysis software	Molecular Devices	MetaXpress	https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metaxpress