Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons

Samenvatting

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)

1. Place the brain tissue in a 100 mm Petri dish and carefully remove the meninges using forceps.
2. Transfer the brain tissue to a 50 mL conical tube and wash it twice with 20 mL of PBS by gently inverting the tube.
3. Incubate the brain tissue in a new 50 mL conical tube by submerging the tissue in cell dissociation solution (see....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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