Plan the number of reaction vials needed for the assay.
TIP: Run each sample in triplicate, including triplicate positive and triplicate negative experimental controls. While running positive and negative sample controls for individual samples is not necessary, it is good practice, especially when performing this assay for the first time. Measure triplicate blanks for each reaction mixture.
Prepare the radiolabeled substrate in advance by aliquoting 0.4 mCi radiolabeled fatty acid per reaction into an Eppendorf tube and dry off the organic phase under nitrogen, air or vacuum.
Add non-radio-labeled substrate/BSA carrier.
TIP: After evaporation, the radiolabeled substrate is in a solid phase attached to the inside of the Eppendorf tube. BSA is added to resolubilize and stabilize the radiolabel in the aqueous solution. Some protocols use alpha-cyclodextrin instead of BSA to resolubilize the radiolabel. Adding a non-radiolabeled substrate to the radiolabeled substrate (i.e. cold + hot) is not necessary, but can increase the signal measured in the assay. Higher substrate levels will increase fatty acid oxidation flux while keeping the radiolabeled signal in the measurable range.
Place solution on thermomixer at 60 C to resuspend radiolabeled substrate.
TIP: Incomplete resuspension of the radiolabeled substrate into the aqueous phase is a common problem with this assay. Using a heated thermomixer set to high-speed shaking can increase the amount of radiolabeled lipid in solution. Prepare this mixture before collecting tissues, which allows 1-2 hours for complete resolubilization.
Prepare reaction mixture from stock solutions for the number of reactions (400 L/reaction +10%) at room temperature with the following final concentration: 100 mM sucrose, 10 mM Tris-HCl, 5 mM KH2PO4, 0.2 mM EDTA, 5 mM NAM, 1 M TSA, 0.3% fatty acid-free BSA, 80 mM KCl, 1 mM MgCl2, 2 mM L-carnitine, 0.1 mM malate, 0.05 mM coenzyme A, and adjust to pH 8.0 (Table 1). This is the entire reaction mixture without DTT, ATP and substrate, which are added immediately before use.
TIP: Prepare the reaction mixture before harvesting mouse tissues. Alternatively, the stock reaction mixture (without DTT, ATP and substrate) can be prepared in advance and stored for up to 6 months at 4 C.
Prepare tissue homogenate as follows8:
a. Prepare sucrose, Tris, EDTA (STE) buffer containing 0.25 M sucrose, 10 mM Tris, 1 mM EDTA (or EGTA), 1 M TSA and 5 mM NAM and place on ice.
TIP: TSA and NAM are added as class I and II, or class III histone deacetylase inhibitors, respectively, and are added only when inhibiting these enzymes is necessary.
b. Anesthetize animal following institutional IACUC protocols.
c. Remove liver, or other tissue of choice.
d. Rinse in cold STE buffer (optional).
e. Homogenize 100-200 mg of liver in 5 volumes STE buffer using a glass 2.0 ml Dounce homogenizer with the "A" pestle (down and up 5 strokes each).
TIP: The "A" pestle is a loose fit and the "B" pestle is a tight fit. Use the "A" pestle to help keep mitochondria intact.
f. Pour homogenate into Eppendorf tubes and place immediately on ice.
g. Continue until all tissues are collected and homogenized.
TIP: No more than 30 min should pass from the time of tissue collection until start of assay, to maintain the integrity of the mitochondria, and ensure a high signal in the assay.
h. Spin crude homogenates at 420 g for 10 min at 4 C.
i. Decant the supernatant containing crude mitochondria into fresh, chilled tubes.
j. Resuspend pellet in STE buffer and spin homogenate again at 420 g for 10 min at 4 C (optional).
k. Discard the pellets and decant supernatant and pool it with supernatant #1.
TIP: Set aside a small volume of tissue homogenate supernatant on ice to measure protein concentration (1:10-1:100 dilution for Bradford/BCA assay).
Dispense 20 l (approximately 250-500 g) of tissue homogenate supernatant into Eppendorf tubes for the reaction on ice
TIP: Highly oxidizing tissues (e.g. brown adipose tissue, liver) requires lower tissue concentrations, whereas low oxidizing tissues (e.g. skeletal muscle, smooth muscle, brain) requires higher tissue concentrations.
a. Include your inhibitor/activator
TIP: The tubes containing inhibitors (e.g. etomoxir) and activators (e.g. AICAR) can be prepared ahead of time, on the same day before tissue collection. Adding the inhibitor/activators to the tissue homogenate supernatant before the buffer and substrate ensures maximal inhibition or activation.
b. Etomoxir irreversibly binds to CPT1 and inhibits mitochondrial oxidation9
c. KCN/NaN3 irreversibly binds to cytochrome oxidase and blocks the electron transport chain6
d. AICAR is an AMP-kinase activator and stimulates oxidation10
Add 2 mM ATP, 1 mM DTT and resuspended radiolabel/BSA to reaction mixture and mix.
TIP: For multiple concentrations of radiolabeled substrate (i.e. dose curve) or various substrates (e.g. palmitate, palmitoleate), split the master mix into different aliquots after DTT and ATP addition, and then add radiolabel/BSA.
Dispense 380 l of reaction mixture into each Eppendorf tube and start the reaction (Figure 2).
Incubate for 30-60 min at 37 C.
TIP: When testing this assay for the first time, try various time points (e.g. fewer than 30 min, greater than 60 min), or different temperatures (e.g. room temperature).
While the reaction is running, prepare new Eppendorf tube reaction collection vials by placing 200 l of 1 M perchloric acid into the tube, and add 10-20 µl of concentrated hyamine hydroxide onto a piece of Whatman filter paper placed in the tube cap.
TIP: Whatman filter paper cut into small discs will fit into cap of an Eppendorf tube. Cut the disc to a size slightly larger than the cap and use the large end of a pipette tip to place the disc in the cap. Making the disc larger than the cap and adding the hyamine hydroxide will help keep the disc in place after inversion (Figure 1).
At the end of the incubation period, transfer the reaction mixture to the Eppendorf collection vials containing perchloric acid.
Quickly close the cap and wait at least 1 h.
TIP: The perchloric acid will precipitate any long-chain (i.e. unoxidized) fatty acids from the reaction mixture. Additionally, CO2 is trapped in the reaction mixture and is liberated upon acidification and captured on the highly basic filter paper. NB: Loss of CO2 in this step was not detected compared to sealed vials.
Open the reaction collection tubes and carefully transfer filter paper disc to a glass scintillation vial.
TIP: Allowing the acid-soluble metabolite and CO2 to equilibrate for more than 1h does not result in greater signal detection, and often causes the paper disc to fall into the acid solution. If this happens, discard the sample and measure the remaining duplicate samples.
Close the reaction collection tubes and spin in microcentrifuge at 14,000 rpm (maximum speed) for 10 min.
Transfer 400 l of the centrifuged reaction mixture containing the acid-soluble metabolites to a scintillation vial.
Add 4 ml of scintillation fluid to all scintillation vials and measure average counts per minute (cpm) over 3 min.
TIP: Include blank vials containing substrate mixture but no tissue homogenate, which is subtracted from the measurement as background. Additionally, to allow for conversion of radioactive counts per minute to fatty acid oxidative activity, measure the radioactive counts per minute of 380 µl of radiolabled reaction mixture. This measurement indicates the amount of signal going into the reaction, and will facilitate the conversion of radioactive counts per minute to fatty acid oxidative activity.
Filter paper counts indicate the amount of CO2 liberated by fatty acid oxidation, while homogenate counts indicate the amount of acid-soluble metabolites (ASM). The radioactive counts in the ASM are typically 5-10-fold higher than in the CO2.