Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons
Published: April 30, 2023
Abstract
Source: Wahlestedt, C., et al., A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons. J. Vis. Exp. (2020)
In this video, we describe a luminescence-based method to assess the neurotoxicity of neurite outgrowth-promoting small molecule compounds on the viability of hNPC-derived neurons. The assay measures intracellular ATP level, an indicator of cell viability, to evaluate neurotoxicity following test compound treatment.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board 1. Isolation and culture of human neural progenitor cells (hNPCs) Place the brain tissue in a 100 mm Petri dish and carefully remove the meninges using forceps. Transfer the brain tissue to a 50 mL conical tube and wash it twice with 20 mL of PBS by gent…
Divulgations
The authors have nothing to disclose.
Materials
| Antibiotic-Antimycotic | Gibco | 15240062 | |
| B27 | Thermo | 17504001 | |
| B27 – minus vitamin A | Thermo | 12587010 | |
| BDNF | PeproTech | 450-02 | |
| BSA | Sigma | A8531 | |
| CellTiter-Glo | Promega | G7572 | |
| CoolCell | Corning | 432000 | Cell freezing containers ensuring standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer |
| CryoStor CS10 | StemCell Technologies | 7930 | Cryopreservation medium containing 10% DMSO |
| DMEM/F12 | Gibco | 11320033 | |
| DMSO | Sigma | 34869-100ML | |
| EGF | Gibco | PHG0311 | |
| FGF | Gibco | PHG6015 | |
| GDNF | PeproTech | 450-10 | |
| Glutamax | Gibco | 35050061 | L-alanyl-L-glutamine supplement |
| Heparin | Calbiochem | 375095 | |
| Laminin | Sigma | L2020-1MG | |
| L-Ascorbic Acid | Sigma | A92902-25G | |
| mFreSR | StemCell Technologies | 5854 | Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells |
| N2 | Gibco | 17502048 | |
| Nunc 384-Well Polystyrene White Microplates | Thermo | 164610 | |
| PBS | Thermo | 10010-049 | |
| Poly-L-lysine | Sigma | P5899-5MG | |
| StemPro Accutase | Gibco | A1110501 | Cell dissociation reagent containing proteolytic and collagenolytic enzymes |
| Neurobasal Medium | Gibco | 21103049 |
Tags
Citer Cet Article
Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons. J. Vis. Exp. (Pending Publication), e21330, doi: (2023).