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

Play Video

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).

View Video