Measurement of Glycolysis and Respiration Using Seahorse
Materials
- Non Buffered DMEM - Sigma cat#D5648 Dissolve one bottle (13.4g) in a litre of water. Adjust pH to 7-7.2 and filter into a sterile bottle.
- FCCP - Prepare as 300 uM stock in DMSO. Need about 20 uL per plate
- Oligomycin - Prepare as a 1 mM Stock in DMSO. Need about 20 uL per plate
- XF24 plate
Protocol
- Email Sydney Bridges to arrange a time to do the experiment. His email is sbridges@med.umich.edu
- Trypsinise cells and resuspend in ~10 mL (for a 10 cm dish).
- Count cells using hemocytometer or cell counter.
- Adjust cell concentration to be 250 000 cells/mL (50K cells/200 uL) using normal growth media
- Add 200 uL of cell suspension per well of a XF24 plate.
- Let cells adhere for > 30min and then add 800 uL of buffer
- Incubate cells overnight.
- Prior to experiment, aspirate 950 uL media then add 1 mL non-buffered DMEM.
- Remove 1 mL media (leaving 50 uL)
- Add 625 uL of non-buffered DMEM for a final volume of 675 uL.
- Bring plate over to Brehm Room 6470 and place in non-CO2 incubator.