ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids

Abstract

Source: Higa, A. et al. High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids. J. Vis. Exp. (2021)

In this video, we demonstrate a rapid, single-addition assay to assess cell viability in patient-derived organoids upon treatment with a test agent using intracellular ATP levels as a marker for cellular metabolic activity. The intracellular ATP levels are measured using the luciferase/luciferin reaction, where the emitted light intensity indicates the cellular viability and, in turn, the test agent's growth-inhibitory potency.

Protocol

1. Growth Inhibition High-Throughput Screening (HTS)

NOTE: The growth inhibitory activity of anticancer agents against patient-derived tumor organoids (PDOs) is evaluated by measuring the intracellular ATP content, as shown in Figure 1. This step is performed using a commercially available cell viability assay kit (see Table of Materials).

1. On day 0, culture the PDOs (e.g., RLUN007) in flasks until adequate numbers of cell clusters are available for the assay. One day before seeding, transfer the PDO suspension from a 75 cm² flask to a 15 mL tube and centrifuge at 200 ....

Results

Figure 1: Summary of the protocol used to create a high-throughput assay system using 384-well microplates.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
384-well Ultra-Low Attachment Spheroid Microplate	Corning	4516	Plates for HTS
Cancer Cell Expansion Media plus	Fujifilm Wako Pure Chemical	032-25745	Medium for F-PDO
CellPet FT	JTEC	-	Cell fragmentation and dispersion equipment
CellTiter-Glo 3D Cell Viability Assay	Promega	G9683	Cell viability luminescent assay, intracellular ATP measuring reagent
Echo 555	Labcyte	-	Liquid handler
EnSpire	PerkinElmer	-	Plate reader
Morphit software, version 6.0	The Edge Software Consultancy		Biological data analysis software
Multidrop Combi	ThermoFisher Scientific	5840300	Cell suspension dispenser
RLUN007	Fujifilm Wako Pure Chemical or Summit Pharmaceuticals International		Lung tumor derived F-PDO
Ultra-Low Attachment 75 cm² Flask	Corning	3814	Culture flask for PDO