Extracellular Flux Assay in 3D Spheroids: A Method for Measuring the Metabolic Activity of Pancreatic Tumor Spheroids
Published: April 30, 2023
Abstract
Source: Noel, P., et al. Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts. J. Vis. Exp. (2017).
This protocol describes the technique for performing extracellular flux assay on 3D spheroids generated from the co-culture of pancreatic cancer cells (Patu8902) and activated pancreatic fibroblasts (PS1) to measure the two major energy-producing pathways, glycolysis and mitochondrial respiration.
Protocol
1. Metabolic Assay of 3D Pancreatic Tumor Spheroids for Bioenergetics Pathways using the Extracellular Flux Analyzer NOTE: In this section, the analysis of the metabolic functions of the spheroids using an extracellular flux analyzer with assay microplates specifically designed for 3D spheroids is described. Washing and Transfer of spheroids from growth plates to spheroid assay microplates. NOTE: Spheroids may be used for me…
Divulgations
The authors have nothing to disclose.
Materials
| 96 well cell repellant plate | USA Scientific/ Griener Bio-One | 655970 | spheroid growth plate |
| D-Glucose | Sigma | D9434 | |
| DPBS | Gibco | 14190-144 | |
| Glycolysis Stress Test Kit | Agilent Technologies | 103020-100 | |
| L-Glutamine | Sigma | 59202C | |
| Mitochondrial Stress Test Kit | Agilent Technologies | 103015-100 | |
| Patu8902 cells | Laboratory Stock | pancreatic ductal adenocarcinoma cells | |
| PS1 cells | Laboratory Stock | activated pancreatic stellate cells | |
| RPMI1640 | Gibco | 11875-093 | growth media for cells, spheroids |
| SeaHorse XFe 96 extracellular flux analyzer | Agilent Technologies | extracellular flux analyzer | |
| Sodium Pyruvate | Sigma | S8636 | |
| XF Base media | Agilent Technologies | 102365-100 | base media for metabolic assays on XFe 96 |
Citer Cet Article
Extracellular Flux Assay in 3D Spheroids: A Method for Measuring the Metabolic Activity of Pancreatic Tumor Spheroids. J. Vis. Exp. (Pending Publication), e20316, doi: (2023).