View source for Seahorse - Mitochondrial Stress Test

[[ Category: Seahorse ]]
[[ Category: Mitochondrial Function ]]
__NOTOC__

==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 
** '''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