9. Different compounds and combinations can be used for bioenergetics experiments depending on the assay type.
10. Ensure the orientation of the cartridge is correct while loading
compounds and when placing inside the machine. Ensure the
lid is removed prior to placing probe or cell plate into the
machine. The sensor cartridge is placed with the bar code
facing the back of the machine and the lot number facing the
front.
11. It is recommended to optimise the FCCP and oligomycin
concentrations for the bioenergetics experiments. In our laboratory, we have determined that 1–1.5 μM is optimal for most
cancer cell lines tested, but this concentration can vary in cell
lines representing different tumour subtypes and types.
12. Avoid MTT, Alamar blue or any other proliferation assay that
measures cellular viability as survival readout of mitochondrial
activity at the end of the assays, because of cellular stress caused
by mitochondrial inhibitors.
13. After performing the analysis in the Seahorse Analyzer, the cell
culture plate can be stored at À80
C and counted later, or
direct cell estimates can be performed on the basis of DNA or
protein concentration-based SRB assay. Normalise the data in
Wave software, a user-friendly software that provides flexibility
in visualisation and data analysis from different assays that the
machine measures, such Mito Stress Test. The generated
report produces an excel file with a separate sheet containing
an explanation of how parameter calculations were performed/
or how the different values of ECAR, OCR or ATP content
measurement were calculated from the assays. ECAR and OCR
measurements following inhibitor treatment are used to calculate fundamental parameters from the Seahorse output including non-mitochondrial oxygen consumption (background),
basal respiration (prior to first injection), maximal respiration
(following uncoupler injection), proton leak, ATP production
(following oligomycin injection) and spare respiratory capacity
(calculated as the difference between basal and maximal respiration). Figure 2 illustrates how these parameters are calculated
from the measurements collected by the Seahorse instrument.
14. Mitochondrial biomass can be affected by your treatments.
This can be accounted for by measuring total mitochondrial
content.
338
Sugandha Bhatia et al.
10. Ensure the orientation of the cartridge is correct while loading
compounds and when placing inside the machine. Ensure the
lid is removed prior to placing probe or cell plate into the
machine. The sensor cartridge is placed with the bar code
facing the back of the machine and the lot number facing the
front.
11. It is recommended to optimise the FCCP and oligomycin
concentrations for the bioenergetics experiments. In our laboratory, we have determined that 1–1.5 μM is optimal for most
cancer cell lines tested, but this concentration can vary in cell
lines representing different tumour subtypes and types.
12. Avoid MTT, Alamar blue or any other proliferation assay that
measures cellular viability as survival readout of mitochondrial
activity at the end of the assays, because of cellular stress caused
by mitochondrial inhibitors.
13. After performing the analysis in the Seahorse Analyzer, the cell
culture plate can be stored at À80
C and counted later, or
direct cell estimates can be performed on the basis of DNA or
protein concentration-based SRB assay. Normalise the data in
Wave software, a user-friendly software that provides flexibility
in visualisation and data analysis from different assays that the
machine measures, such Mito Stress Test. The generated
report produces an excel file with a separate sheet containing
an explanation of how parameter calculations were performed/
or how the different values of ECAR, OCR or ATP content
measurement were calculated from the assays. ECAR and OCR
measurements following inhibitor treatment are used to calculate fundamental parameters from the Seahorse output including non-mitochondrial oxygen consumption (background),
basal respiration (prior to first injection), maximal respiration
(following uncoupler injection), proton leak, ATP production
(following oligomycin injection) and spare respiratory capacity
(calculated as the difference between basal and maximal respiration). Figure 2 illustrates how these parameters are calculated
from the measurements collected by the Seahorse instrument.
14. Mitochondrial biomass can be affected by your treatments.
This can be accounted for by measuring total mitochondrial
content.
338
Sugandha Bhatia et al.