9. Microplate reader.
10. High-content microscopy imaging system (e.g. InCell
Analyzer 2200).
11. Flow cytometer.
2.2 Reagents
and Solutions (96-Well
Assay Format)
1. Seahorse XFe96 FluxPak containing probe plate, 96-well tissue
culture plate and calibrant (Seahorse Bioscience, 102601-100).
2. Seahorse XF Base Medium (DMEM or RPMI without phenol
red) or other medium (without glucose, glutamine, sodium
bicarbonate, sodium pyruvate, phenol red).
3. Substrate Supplements (final concentration): 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM glucose.
4. Seahorse XF Cell Mito Stress Kit (Seahorse Bioscience,
103015-100) containing oligomycin, FCCP and Rotenone/
Antimycin A. Aliquots are dissolved in media to 100 mM stock
concentrations.
5. CellTak™ (Corning, Catalog# 354240), collagen, polyornithine or poly-L-Lysine.
6. Hoechst nuclear dye.
7. Syringe Filters, 0.22 μM pore size, 4 mm membrane diameter.
8. Acids and Bases for adjusting pH.
9. Media reservoirs for multichannel dispensing.
10. DNA or protein-based cell counting kits.
11. Trypsin/EDTA Solution.
12. Accutase.
13. Dimethylsulfoxide (DMSO).
14. Fetal Bovine Serum (FBS).
15. Mitotracker dyes (mitochondrial mass and mitochondrial
membrane potential).
16. FACS sorting buffer (1Â PBS with 0.5% FBS, 1 mM EDTA,
25 mM HEPES pH 7).
3 Methods
3.1 Methodology
Overview
The Seahorse assay is performed on live cells. Cells are seeded into
specialised 96-well cell culture-treated plates, supplied by the manufacturer. The wells are funnel-shaped which allows the probes to
create a snug microchamber of ~2 μL immediately above the cells to
allow highly sensitive measurement of dissolved H
+ and O 2 . The
cells are allowed to adhere, typically overnight. On the day of the
assay, the cells are transferred to an unbuffered media with fixed pH
equilibrated at temperature and in atmospheric oxygen. During the
330
Sugandha Bhatia et al.
10. High-content microscopy imaging system (e.g. InCell
Analyzer 2200).
11. Flow cytometer.
2.2 Reagents
and Solutions (96-Well
Assay Format)
1. Seahorse XFe96 FluxPak containing probe plate, 96-well tissue
culture plate and calibrant (Seahorse Bioscience, 102601-100).
2. Seahorse XF Base Medium (DMEM or RPMI without phenol
red) or other medium (without glucose, glutamine, sodium
bicarbonate, sodium pyruvate, phenol red).
3. Substrate Supplements (final concentration): 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM glucose.
4. Seahorse XF Cell Mito Stress Kit (Seahorse Bioscience,
103015-100) containing oligomycin, FCCP and Rotenone/
Antimycin A. Aliquots are dissolved in media to 100 mM stock
concentrations.
5. CellTak™ (Corning, Catalog# 354240), collagen, polyornithine or poly-L-Lysine.
6. Hoechst nuclear dye.
7. Syringe Filters, 0.22 μM pore size, 4 mm membrane diameter.
8. Acids and Bases for adjusting pH.
9. Media reservoirs for multichannel dispensing.
10. DNA or protein-based cell counting kits.
11. Trypsin/EDTA Solution.
12. Accutase.
13. Dimethylsulfoxide (DMSO).
14. Fetal Bovine Serum (FBS).
15. Mitotracker dyes (mitochondrial mass and mitochondrial
membrane potential).
16. FACS sorting buffer (1Â PBS with 0.5% FBS, 1 mM EDTA,
25 mM HEPES pH 7).
3 Methods
3.1 Methodology
Overview
The Seahorse assay is performed on live cells. Cells are seeded into
specialised 96-well cell culture-treated plates, supplied by the manufacturer. The wells are funnel-shaped which allows the probes to
create a snug microchamber of ~2 μL immediately above the cells to
allow highly sensitive measurement of dissolved H
+ and O 2 . The
cells are allowed to adhere, typically overnight. On the day of the
assay, the cells are transferred to an unbuffered media with fixed pH
equilibrated at temperature and in atmospheric oxygen. During the
330
Sugandha Bhatia et al.