Luminescence-Based Assay to Assess Neurotoxicity of Test Compounds on Neurons
Transcript
Neurite outgrowth — where neurons develop projections — is crucial for neural function and regeneration.
To assess the neurotoxic effects of neurite outgrowth-promoting test compounds in vitro, obtain a human neural progenitor cell, hNPC, suspension in differentiation media.
Seed the cells into multi-well plate wells coated with poly-L-lysine and laminin, facilitating cell attachment to the well bottom and supporting cellular growth. The differentiation media constituents promote hNPC differentiation into neurons.
Treat one set of wells with neurite outgrowth-promoting test compound. This interacts with the hNPC-derived neurons and may induce neurite outgrowth, without disrupting the cellular metabolic processes and subsequent concentration of adenosine triphosphate, ATP — the primary cellular energy source.
Add the single-step reagent containing detergents, luciferase enzyme, and luciferase substrate, luciferin, and ATPase inhibitors along with magnesium ions, and mix.
Detergents disrupt the cellular membrane, resulting in cell lysis and intracellular ATP release into the solution. ATPase inhibitors inhibit the released endogenous ATPase, which may interfere with ATP measurement. Centrifuge, sedimenting cell debris.
During incubation, the released ATP with magnesium ions cause luciferin to bind to luciferase, resulting in luciferin oxidation into highly-excited oxyluciferin, emitting a bioluminescent photon upon relaxation to its ground state. Using a microplate reader, measure the luminescence, indicative of ATP concentration.
Higher ATP concentration represents higher cell viability, suggesting no significant test compound toxicity on neurons.
