This study investigates the neurotoxic effects of neurite outgrowth-promoting compounds on human neural progenitor cells (hNPCs). The methodology includes assessing ATP concentration as an indicator of cell viability following treatment.
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.
To coat the plate, add 30 microliters of poly-L-lysine into each well of a 384-well plate, and incubate the plate at room temperature for 1 hour. Wash it twice with PBS, and let it dry for approximately 30 minutes.
Add 30 microliters of laminin to each well, then, incubate the plate at 37 degrees Celsius for 2 hours. Repeat the washes with PBS, and proceed with plating the cells. Add 20,000 single-cell neurospheres in 25 microliters of differentiation media, to each well of the 384-well plate, and incubate the plate at 37 degrees Celsius for 5 days.
High-precision pipetting skills are required for plating the exact number of neurospheres that are disaggregated into single cells. Therefore, differentiating the neurospheres into neurons, before plating is recommended to achieve more reproducible results.
After the incubation, add 5 microliters of test compound to each well at 6 times the desired concentration, and incubate the cells for another 24 hours.
To perform the cell viability assay, add 30 microliters of luminescent reagent to each well, and leave the plate on a shaker for 2 minutes. Centrifuge the plate at 300 to 400 times g for 30 seconds. Then, incubate the plate at room temperature for 10 minutes, protecting it from light. After the incubation, measure the luminescence on a microplate reader.
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