ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids
Published: April 30, 2023
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). On day 0, culture the PDOs (e.g., RLUN007) in flasks until adequate numbers of cell clusters …
Representative Results
Figure 1: Summary of the protocol used to create a high-throughput assay system using 384-well microplates.
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids. J. Vis. Exp. (Pending Publication), e21218, doi: (2023).