Seahorse XF Media

This minimal media stock is the base media for both the glycolysis and mitochondrial stress assays. It contains no buffering and no carbon sources. It is a non-buffered media but still needs to be brought to pH 7.4 and checked before each assay.

For glycolysis assays add to warmed XF base media just before the assay and recheck pH:

• 1-4 mM L-Glutamine (Generally 2 mM is 1mL/100mL of 200 mM stock)

For mitochondrial assays and cellular phenotype assays add to warmed XF base media just before the assay and recheck pH:

• 0.5-10 mM Sodium pyruvate (Generally 1 mM is 100uL/100mL of 1M stock)
• 1-4 mM L-Glutamine (Generally 2 mM is 1mL/100mL of 200 mM stock)
• 2-25 mM Glucose (Generally 10 mM is 1mL/100mL of 1M stock)

To make base media add the following:

Reagent	Stock Concentration	Volume (500 mL)	Final Concentration
Magnesium sulfate	1 M	400 uL	0.8 mM
Calcium chloride	1 M	900 uL	1.8 mM
Sodium Chloride	4 M	17.8 mL	143 mM
Potassium chloride	1 M	2.7 mL	5.4 mM
Sodium phosphate	200 mM	2.28 mL	0.91 mM

Add ~450 mL water, bring pH to 7.4 carefully (there is very little buffering capacity, so add NaOH slowly). Bring final volume up to 500 mL and sterile filter into an autoclaved bottle. Store at 4C.