This study investigates the role of adenosine triphosphate (ATP) as a marker for cell viability in tumor organoids. By measuring ATP levels, researchers can assess the growth-inhibitory potential of test compounds on tumor cells.
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.
After 24 hours of medium change, mince the tumor organoids using a cell fragmentation and dispersion instrument, equipped with a filter holder containing a 70-micrometer mesh filter. Dilute 15 millimeters of tumor organoid suspension 10 times.
Use the cell suspension dispenser to seed 40 microliters of diluted tumor organoid suspension in a 384-well ultra-low attachment spheroid microplate.
After 24 hours, use a liquid handler to add 0.04 microliters of test agent solutions from 10 serial dilutions at final concentration ranges of 20 micromolar to 1.0 nanomolar in the wells, and incubate the plate for 6 days.
At the end of the incubation, add intracellular ATP-measuring reagent in the test wells. Use a mixer to mix the contents of the plate and incubate the plate for 10 minutes at approximately 25 degrees Celsius. Use a plate reader to measure the intracellular ATP content as luminescence.
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