An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms
Published: April 30, 2023
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 Treatment of C. elegans with test samples 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. Seal the plate with parafilm and incubate at 15 °C and 120 rpm for 3 days. <…
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms. J. Vis. Exp. (Pending Publication), e21285, doi: (2023).