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.

Protocol

The Day Before the Assay

• 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).

The Day of the Assay

• 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. Will add 56 uL to each port
  • Tube B 18 uL FCCP. Will add 62 uL to each port
  • Tube C 9 uL Rotenone and 9 uL Antimycin A. Will add 69 uL to each port
• 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