Seahorse - Mitochondrial Stress Test - Revision history

Davebrid: /* The Day of the Assay */ clarified what is being added to ports

2025-09-02T18:06:32Z

The Day of the Assay: clarified what is being added to ports

← Older revision		Revision as of 18:06, 2 September 2025
Line 28:		Line 28:
	* 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. Will add 56 uL		** '''Tube A''' 18 uL Oligomycin. Will add 56 uL to each port
	** '''Tube B''' 18 uL FCCP. Will add 62 uL		** '''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		** '''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.'''		* 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

Davebrid: /* The Day of the Assay */ Updated with volumes of reagents

2025-08-12T19:07:25Z

The Day of the Assay: Updated with volumes of reagents

← Older revision		Revision as of 19:07, 12 August 2025
Line 28:		Line 28:
	* 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

Davebrid: removed table of contents

2017-07-31T12:28:38Z

removed table of contents

← Older revision		Revision as of 12:28, 31 July 2017
Line 1:		Line 1:
	[[ Category: Seahorse ]]		[[ Category: Seahorse ]]
	[[ Category: Mitochondrial Function ]]		[[ Category: Mitochondrial Function ]]
			__NOTOC__

	==Materials==		==Materials==

Snyderds: /* Protocol */ added "remove lid" in 2nd to last step

2017-06-14T17:02:55Z

Protocol: added "remove lid" in 2nd to last step

← Older revision		Revision as of 17:02, 14 June 2017
Line 32:		Line 32:
	* 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. '''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

Snyderds at 13:39, 14 June 2017

2017-06-14T13:39:11Z

← Older revision		Revision as of 13:39, 14 June 2017
Line 24:		Line 24:
	*** 1 mM Pyruvate (100 uL of a 1M stock)		*** 1 mM Pyruvate (100 uL of a 1M stock)
	** Re-adjust the pH to 7.4 (7.35-7.45)		** Re-adjust the pH to 7.4 (7.35-7.45)
	* Wash cells twice with 500 mL of pre-warmed media, add 500 uL final volume to each well		* 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		* 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)

Davebrid: Wrote initial page for seahorse assay

2017-05-22T20:06:25Z

Wrote initial page for seahorse assay

← Older revision		Revision as of 20:06, 22 May 2017
Line 1:		Line 1:
	[[ Category: Seahorse ]]		[[ Category: Seahorse ]]
	[[ Category: Mitochondrial Function ]]		[[ Category: Mitochondrial Function ]]

			==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 mL 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. '''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

Davebrid: created placeholder page

2017-05-16T19:33:10Z

created placeholder page

New page

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