ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids
Transkript
Adenosine triphosphate, ATP, the primary cell energy source, is vital for diverse cellular functions. Under stress, intracellular ATP levels are negatively impacted, making ATP a good marker for metabolically active, viable cells.
To begin, seed tumor cell suspension into a black-walled, clear-bottom microplate for minimal light scattering during light intensity measurement. The microplate consists of a hydrogel coating that prevents cells from adhering to the plate, enabling cell-to-cell interaction and self-assembly into tumor organoids – three-dimensional tissue cultures that mimic tumor microenvironment.
Next, treat with a test compound. The test compound interacts with the organoid cells and, depending on its growth-inhibitory potential, may disrupt the energy-producing metabolic processes, resulting in intracellular ATP depletion.
Post-treatment, add a one-step reagent comprising detergents, ATPase inhibitors, the enzyme luciferase, luciferase-specific substrate, luciferin, and magnesium ions. Incubate for the desired duration.
The detergent solubilizes the cellular membranes, causing cell lysis and ATP extraction into solution, while the ATPase inhibitors inactivate any released endogenous ATPases, stabilizing ATP.
Following its extraction, ATP, in the presence of magnesium ions, facilitates luciferin to bind to luciferase, which oxidizes luciferin into high-energy oxyluciferin that upon relaxation, generates a bioluminescent photon.
Using a luminometer, measure the light intensity produced by the photons, representative of intracellular ATP concentration. Lower amounts of ATP indicate lower cell viability, suggesting higher growth-inhibitory potential of the test compound.
