container. Add 1.2 g of tris(hydroxymethyl)aminomethane and
190 mg of EGTA. Mix and adjust pH to 7.2 with HCl. Make
up to 500 ml with mQ water and store at 4C (weeks) or
20 C (months). On the day of use, add 500μl of bovine
serum albumin solution (final concentration of 0.15% BSA)
and 25μl of inorganic phosphate 1 M (final concentration of
5mMPi-Tris) for 50 ml of KCl buffer.- XF assay medium. Add 0.5 ml ofD-glucose 1 M to 49.5 ml of
M199 medium. - 5% FCS (v/v)/XF assay medium. Add 0.5 ml ofD-glucose 1 M
and 2.5 ml of heat-inactivated FCS to 47 ml of M199 medium. - Ethanol: 70% (v/v). Add 700 ml of ethanol absolute in a glass
cylinder and complete to 1 L with mQ water. - Ethanol: 30% (v/v). Add 300 ml of ethanol absolute in a glass
cylinder and complete to 1 L with mQ water.
2.4 Substrates
and Inhibitors
- Tris-buffer: 50 mM Tris. Add ~50 ml of mQ water to a glass
beaker containing a magnet. Weigh 605.7 mg of Tris. Mix and
adjust pH to 7.4 with HCl 1 N. Make up to 100 ml with mQ
water, aliquot and store at 20 C. - Glutamate/malate: 0.5 M glutamate/0.25 M malate. Dissolve
422.7 mg ofL-glutamic acid sodium salt and 222.6 mg ofL-
malic acid disodium salt in 5 ml of Tris-buffer 50 mM. Aliquot
(250μl) in 0.5-ml tubes and store at 20 C. - Succinate/malate: 0.5 M succinate/0.25 M malate. Dissolve
675 mg of succinate disodium salt hexahydrate and 222.6 mg
ofL-malic acid disodium salt in 5 ml of Tris-buffer 50 mM.
Aliquot (250μl) in 0.5 ml tubes and store at 20 C. - Malate: 0.25 M malate. Dissolve 222.6 mg ofL-malic acid
disodium salt in 5 ml of Tris-buffer 50 mM. Aliquot (250μl)
in 0.5 ml tubes and store at 20 C. - Palmitoyl-DL-carnitine: 20 mM palmitoyl-DL-carnitine. Dis-
solve 43.6 mg of palmitoyl-DL-carnitine chloride in 5 ml of
mQ water. Aliquot (250μl) in 0.5 ml tubes and store at
20 C. - Rotenone: 2 mM rotenone. Dissolve 3.95 mg of rotenone in
5 ml of ethanol absolute. Aliquot (100μl) in 0.5 ml tubes and
store at 20 C. - ADP: 100 mM ADP. Dissolve 213.6 mg of adenosine 5-
(^0) -diphosphate sodium salt in 5 ml of Tris-buffer 50 mM. Ali-
quot (200μl) in 0.5 ml tubes and store at 20 C.
- Oligomycin: 1 mg/ml oligomycin. Dissolve 2 mg of oligomy-
cin in 2 ml of DMSO. Aliquot (100μl) in 0.5 ml tubes and
store at 20 C.
Methods for Assessing Mitochondrial OXPHOS 277