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.
Divulgaciones
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
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).