Extracellular Flux Assay in 3D Spheroids: A Method for Measuring the Metabolic Activity of Pancreatic Tumor Spheroids

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.

1. Washing and Transfer of spheroids from growth plates to spheroid assay microplates.
   NOTE: Spheroids may be used for metabolic assays when they reach a diameter of ~500 µm.
   1. The day prior to performing the assay, hydrate the probe or sensor cartridge wi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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