Seahorse - Mitochondrial Stress Test

Materials

Seahorse XF Media
XF Cartridge
Cells in XF24 plate, either grown in the plate or seeded at a previously established density where the OCR is 40-500 mL/min.
Injection stock solutions (1 mM Oligomycin, 1 mM FCCP, 1 mM Rotenone and 1 mM Antimycin A) made up in DMSO at 1000X. Aliquots in the -20 in Seahorse Reagents box.
Check if the machine is free.

Hydrate a cartridge by placing 1 mL/well of XF Calibration Media into each well of a XF24 cartridge. Place in non-CO2, humidified, 37C incubator overnight to hydrate.
Ensure that there is sufficient XF media and stock solutions.
Prepare cells if necessary, leave in CO2 incubator overnight. Make sure to leave blank wells (typically A5, B3, C4, D2).

Prepare media by the following steps:
- Place 100mL in non-CO2, humidified, 37C incubator for ~30 minutes to warm up.
- Add the following solutions (these are standard concentrations and may be slightly different for your cell line of interest):
  - 10 mM Glucose (1 mL of a 1M stock into 100 mL)
  - 2 mM Glutamine (1 mL of a 200 mM stock)
  - 1 mM Pyruvate (100 uL of a 1M stock)
- Re-adjust the pH to 7.4 (7.35-7.45)
Wash cells twice with 500 uL of pre-warmed media, add 500 uL final volume to each well
Incubate for 1h in non-CO2, humidified, 37C incubator to get rid of any extra CO2
While plate is incubating, prepare injection solutions in 1.8 mL of prepared XF Media. These are standard concentrations, and should be optimized for your system (especially FCCP)
- Tube A 18 uL Oligomycin
- Tube B 18 uL FCCP
- Tube C 9 uL Rotenone and 9 uL Antimycin A
Add the compounds to the injection ports in the cartridge (A, bottom right; B bottom left; C top right). Make sure the barcode is on the right hand side.
Set up your protocol, editing a similar template and saving with the date and experiment type
Start the protocol, inserting the cartridge with the lid removed. Make sure the barcode is on the right hand side.
Once the cartridge is calibrated insert the cell plate at the prompt. If the calibration fails, abort the run and try the calibration again