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

1. Email Sydney Bridges to arrange a time to do the experiment. His email is sbridges@med.umich.edu
2. Trypsinise cells and resuspend in ~10 mL (for a 10 cm dish).
3. Count cells using hemocytometer or cell counter.
4. Adjust cell concentration to be 250 000 cells/mL (50K cells/200 uL) using normal growth media
5. Add 200 uL of cell suspension per well of a XF24 plate.
6. Let cells adhere for > 30min and then add 800 uL of buffer
7. Incubate cells overnight.
8. Prior to experiment, aspirate 950 uL media then add 1 mL non-buffered DMEM.
9. Remove 1 mL media (leaving 50 uL)
10. Add 625 uL of non-buffered DMEM for a final volume of 675 uL.
11. Bring plate over to Brehm Room 6470 and place in non-CO2 incubator.