Seahorse - Mitochondrial Stress Test: Difference between revisions
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→The Day of the Assay: Updated with volumes of reagents |
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* Incubate for 1h in non-CO2, humidified, 37C incubator to get rid of any extra CO2 | * 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) | * 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 A''' 18 uL Oligomycin. Will add 56 uL | ||
** '''Tube B''' 18 uL FCCP | ** '''Tube B''' 18 uL FCCP. Will add 62 uL | ||
** '''Tube C''' 9 uL Rotenone and 9 uL Antimycin A | ** '''Tube C''' 9 uL Rotenone and 9 uL Antimycin A. Will add 69 uL | ||
* 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.''' | * 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 | * 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.''' | * 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 | * Once the cartridge is calibrated insert the cell plate at the prompt. If the calibration fails, abort the run and try the calibration again | ||