2. Lift the cell lines with Accutase and incubate 5 Â 10
5 cells with
mitochondrial markers in media for 1 h at 37
C.
3. Wash the cells once and resuspend in sorting buffer.
4. Strain the samples using cell strainer into 5 mL propylene
tubes.
5. Perform quantitative fluorescence analysis using a suitable flow
cytometer (see Note 14).
3.5 Analysis
1. The mitochondrial respiration and extracellular acidification
rate, normalised to cell number, are plotted against time and
show the metabolic response following the addition of inhibitors (see Fig. 3a, b for example).
2. The bioenergetic values of ECAR and OCR can be plotted
together for a comparative view. This view can compare intraexperimental phenotypes with low metabolic or dormant cells
in the lower left quadrant, more glycolytic cells in bottom right
quadrant, more OXPHOS-dependent cells in the top left quadrant and cells utilising both pathways in the most metabolic top
right quadrant. See Fig. 3c for an example, where the results
from an MET model cell line have been summarised.
4 Notes
1. Studies can be performed under hypoxia, but as the Seahorse
undertakes measurements under ‘atmospheric’ oxygen, the
Seahorse machine must be placed inside a hypoxia hood.
2. The mixing and movement of the Seahorse probes can disturb
cells that are weakly adherent. To avoid this, plates should be
precoated to improve cell adherence during the assay. The
manufacturer recommends CellTak™ for this. Alternatively,
substitutes of ECM matrix, such as collagen, poly-ornithine
or poly-L-Lysine can be used. In our experience, coating also
improves even cell distribution when plating, and better adherence of cells during media exchanges prior to and during the
assay.
3. The 4 corner wells of the 96-well plate (A1, A12, H1 and H12)
are temperature correction wells and must not have cells seeded
in them. Treat these wells as media blanks.
4. It is recommended to perform a pilot study to assess metabolic
potential for range of cell densities, for optimal concentration
of inhibitors of oligomycin and FCCP across different cancer
cell lines. In our experience we have found a minimum of
4 replicate wells to be required for cell-line observations.
336
Sugandha Bhatia et al.
5 cells with
mitochondrial markers in media for 1 h at 37
C.
3. Wash the cells once and resuspend in sorting buffer.
4. Strain the samples using cell strainer into 5 mL propylene
tubes.
5. Perform quantitative fluorescence analysis using a suitable flow
cytometer (see Note 14).
3.5 Analysis
1. The mitochondrial respiration and extracellular acidification
rate, normalised to cell number, are plotted against time and
show the metabolic response following the addition of inhibitors (see Fig. 3a, b for example).
2. The bioenergetic values of ECAR and OCR can be plotted
together for a comparative view. This view can compare intraexperimental phenotypes with low metabolic or dormant cells
in the lower left quadrant, more glycolytic cells in bottom right
quadrant, more OXPHOS-dependent cells in the top left quadrant and cells utilising both pathways in the most metabolic top
right quadrant. See Fig. 3c for an example, where the results
from an MET model cell line have been summarised.
4 Notes
1. Studies can be performed under hypoxia, but as the Seahorse
undertakes measurements under ‘atmospheric’ oxygen, the
Seahorse machine must be placed inside a hypoxia hood.
2. The mixing and movement of the Seahorse probes can disturb
cells that are weakly adherent. To avoid this, plates should be
precoated to improve cell adherence during the assay. The
manufacturer recommends CellTak™ for this. Alternatively,
substitutes of ECM matrix, such as collagen, poly-ornithine
or poly-L-Lysine can be used. In our experience, coating also
improves even cell distribution when plating, and better adherence of cells during media exchanges prior to and during the
assay.
3. The 4 corner wells of the 96-well plate (A1, A12, H1 and H12)
are temperature correction wells and must not have cells seeded
in them. Treat these wells as media blanks.
4. It is recommended to perform a pilot study to assess metabolic
potential for range of cell densities, for optimal concentration
of inhibitors of oligomycin and FCCP across different cancer
cell lines. In our experience we have found a minimum of
4 replicate wells to be required for cell-line observations.
336
Sugandha Bhatia et al.