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.
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)
2. Passaging the hNPCs
3. Freezing the hNPCs
4. Neurotoxicity assessment
NOTE: Cytotoxicity of test compounds is evaluated in 384-well plates (see Table of Materials) using a luminescent cell viability assay (see Table of Materials). The hNPCs are prepared following the same method, except for slight modifications, described in the “Differentiation and Treatment of hNPCs” section. Subsequently, a luminescent signal generated by luminescent cell viability assay is measured utilizing a microplate reader. The luminescent signal is proportional to the cellular ATP concentration, which itself is directly proportional to the number of viable cells present in each well.
Table 1. Components required for making 100 mL of culture media
Amount | Component |
100 µL | EGF (20 ng/mL) |
100 µL | FGF (10 ng/mL) |
2 mL | B-27, Minus vitamin A (50X) |
1 mL | L-alanyl-L-glutamine (100X) (see table of materials) |
4 µL | Heparin (2 μg/mL) |
96.8 mL | Neuronal cell culture medium (see table of materials) |
The authors have nothing to disclose.
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 |