creating a microchamber immediately above the cells. The probe
simultaneously monitors extracellular acidification rate (ECAR) by
measuring the H
+ in the media, which represents lactate production
from glycolysis and the oxygen consumption rate (OCR), by measuring dissolved oxygen which reflects mitochondrial respiration.
These key parameters can be used to determine rates of respiration
and glycolysis (e.g. Mito Stress Test and Glycolysis Stress Test), the
relative contribution of each pathway to total energy production
(e.g. ATP Rate Assay and Glycolytic Rate Assay) and the preference
of cells for different fuels or substrates such as glucose, glutamine or
fatty acids (e.g. Fuel Flex Assay). These assays measure mitochondrial and glycolytic function by the sequential injection of metabolic inhibitors. Here we describe the utilisation of the Mito Stress
Test to determine the relative function and dependency of cells with
different metastatic potential on OXPHOS and glycolysis. The ATP
Rate Assay may also be recommended for this type of analysis.
Until recently, studies have shown that decreased mitochondrial respiration and increased glycolytic activity, associated with
EMT and metastasis, may be context-dependent [22]. The concept
of a hybrid metabolic phenotype is now evolving, for example
metastatic breast cancer cell lines 66 cl4 and 4 T1 have both
enhanced ECAR and OCR compared with non-metastatic 67NR
cells [23]. Studying metabolism in models of EMP can highlight
unique or essential attributes that satisfy energy demands and fuel
utilisation to support migration and invasion capabilities, and heralds a major advance in our understanding of the role for metabolic
reprogramming in changing cell phenotypes. Additionally, studies
of bioenergetic flux of cancer cells can be useful in assessing metabolic drugs that may impact EMP status [24]. Thus, this technology can generate new insights into the dynamics of cancer cell
metabolism within the EMP spectrum.
2 Materials
2.1 Equipment
1. Seahorse XF
e 96 Extracellular Flux Analyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
2. CO 2 incubator for growing cells in an atmosphere of 5–10%
CO 2 (see Note 1).
3. Non-CO 2 incubator to stabilise the cells in atmospheric oxygen immediately prior to analysis.
4. Laminar flow hood.
5. Cell counter.
6. Single channel and multichannel dispensing pipette.
7. pH meter.
8. Water bath.
Studying EMP Bioenergetic Switch Using Seahorse Analyzer
329
simultaneously monitors extracellular acidification rate (ECAR) by
measuring the H
+ in the media, which represents lactate production
from glycolysis and the oxygen consumption rate (OCR), by measuring dissolved oxygen which reflects mitochondrial respiration.
These key parameters can be used to determine rates of respiration
and glycolysis (e.g. Mito Stress Test and Glycolysis Stress Test), the
relative contribution of each pathway to total energy production
(e.g. ATP Rate Assay and Glycolytic Rate Assay) and the preference
of cells for different fuels or substrates such as glucose, glutamine or
fatty acids (e.g. Fuel Flex Assay). These assays measure mitochondrial and glycolytic function by the sequential injection of metabolic inhibitors. Here we describe the utilisation of the Mito Stress
Test to determine the relative function and dependency of cells with
different metastatic potential on OXPHOS and glycolysis. The ATP
Rate Assay may also be recommended for this type of analysis.
Until recently, studies have shown that decreased mitochondrial respiration and increased glycolytic activity, associated with
EMT and metastasis, may be context-dependent [22]. The concept
of a hybrid metabolic phenotype is now evolving, for example
metastatic breast cancer cell lines 66 cl4 and 4 T1 have both
enhanced ECAR and OCR compared with non-metastatic 67NR
cells [23]. Studying metabolism in models of EMP can highlight
unique or essential attributes that satisfy energy demands and fuel
utilisation to support migration and invasion capabilities, and heralds a major advance in our understanding of the role for metabolic
reprogramming in changing cell phenotypes. Additionally, studies
of bioenergetic flux of cancer cells can be useful in assessing metabolic drugs that may impact EMP status [24]. Thus, this technology can generate new insights into the dynamics of cancer cell
metabolism within the EMP spectrum.
2 Materials
2.1 Equipment
1. Seahorse XF
e 96 Extracellular Flux Analyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
2. CO 2 incubator for growing cells in an atmosphere of 5–10%
CO 2 (see Note 1).
3. Non-CO 2 incubator to stabilise the cells in atmospheric oxygen immediately prior to analysis.
4. Laminar flow hood.
5. Cell counter.
6. Single channel and multichannel dispensing pipette.
7. pH meter.
8. Water bath.
Studying EMP Bioenergetic Switch Using Seahorse Analyzer
329