Cardiomyocyte fatty acid oxidation assay

Material:

24-well plates

35 mm petri dishes

Bovine Serum Albumin heat shock fraction, protease free, fatty acid free (Merck Cat# A7030)

Dulbecco′s Modified Eagle′s Medium - low glucose (Merck Cat# D6046)

Falcon® 70 µm Cell Strainer (Corning Cat# 352350)

Fetal Bovine Serum, heat inactivated, certified, One Shot™ (ThermoFisher Scientific Cat# A3840001)

Hepes (Merck Cat# 3375)

L-Carnitine (Cayman Chemical Cat# 21489)

MicroAmp™ Clear Adhesive Film (ThermoFisher Scientific Cat# 4306311)

Oleic acid (Merck Cat# O1383)

Oleic Acid, [1-14C]-, 50µCi (1.85MBq) (PerkinElmer Cat# NEC317050UC)

Penicillin-Streptomycin (Merck Cat# P4333)

Pierce™ Primary Cardiomyocyte Isolation Kit (ThermoFisher Scientific Cat# 88281)

Sterile 0.5-, 1.5-, 15- and 50-ml tubes

Trypan blue stain 0.4% (Invitrogen Cat# T10282)

Equipment:

Cell incubator

Countess 3 Automated Cell Counter

Fume hood

Micro-centrifuge

TRI-CARB 4810TR 110 V Liquid Scintillation Counter

Step 1: Cardiomyocyte isolation

1. Prepare isolation medium (DMEM medium containing 10% heat inactivated FBS and 1% penicillin/streptomycin):
   1. 445 ml DMEM for Primary Cell Isolation (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer)
   2. 50 ml Heat inactivated FBS
   3. 5 ml Penicillin/Streptomycin solution
2. Immerse excised foetal hearts (up to 2 hearts) in 500 µl HBSS (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer) in a 1.5-ml tube.
3. Mince hearts into small fragments and wash them twice with 500 μl ice-cold HBSS. Wait a minute for the tissue to precipitate between washes (leave tubes on ice).
4. Aspirate the HBSS and add 0.2 mL Cardiomyocyte Isolation Enzyme 1 (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer) and 10 μl Cardiomyocyte Isolation Enzyme 2 (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer)
5. Mix gently by flicking the tube and incubate in water bath at 37 °C for 30 minutes.
6. Remove the enzyme solution and wash tissue twice with 500 μl ice-cold HBSS. ATTENTION: the tissue is now digested and can be easily aspirated with the buffer. Proceed with caution.
7. Add 0.5 mL complete DMEM for Primary Cell Isolation. Pipette up and down 25-30 times using a p1000 pipette.
8. Strain cells using a 70 µm strainer and wash strainer with 2 ml medium.
9. Transfer cells to a 35 mm petri dish and let them incubate at 20 % O₂ for 15 minutes for fibroblast adhesion.
10. After fibroblast adhesion, collect the medium with unattached cells into a fresh 15 ml tube and mix 10 μL single-cell suspension with 10 μL 0.4% trypan blue in a new 0.5-mL microcentrifuge tube.
11. Transfer 12 μl trypan blue-stained cell suspension to a counting chamber.
12. Using a Countess system, count the total number of live cells. 
13. Transfer 1 x 10⁵ cells to a well on a 24 well/plate.

Step 2: Measurement of fatty acid oxidation 

1. Prepare the FAO medium as follows: 
   1. Calculate the amount of radiolabelled oleate required. Each sample is assayed in quadruplicate + an extra reaction for total counts + blank.
   2. Prepare FAO medium (DMEM medium containing 12.5mM HEPES, 0.3% BSA, 1 mM L-Carnitine and 100 µM unlabelled oleate):
      1.  467.8 ml DMEM low glucose
      2.  5 ul 1.25 M HEPES
      3.  16.7 ul 9% fatty acid-free BSA
      4.  10 ul 50 mM L-Carnitine
      5.  0.5 ul 100 mM Oleate
   3. Transfer 1 μCi [1-14C]-oleate per reaction (usually 1 ul per reaction) to a 0.5-ml tube and let the ethanol evaporate inside a fume hood.
   4. Resuspend the radionuclide into the FAO medium and incubate it at 37 ºC in water bath for 30 minutes.
2. Prior to adding the FAO medium into the wells, attach the filter paper disc to the inside of the plate adhesive film (it will glue nicely) and pipette 25 μL of 1 M NaOH on it.
3. Aspirate the media from the wells, wash cells once with warm PBS and add 500 μl of FAO medium.
4. Also pipette 500 μl FAO medium into an empty well to have a background count check.
5. Seal the plate with the plate film and place it in an incubator at 37°C for 3h. ATTENTION: Make sure all the wells in use are properly sealed by manually pressing the film against the top of the walls and rubbing lightly.
6. After 3 hours, bring the plate into a fume hood and carefully remove the plate film. ATTENTION: Avoid disturbing the paper disc while removing the film, which could lead it to detach and fall into the medium.
7. Add 200 ul of 37% hydrochloric acid into the well and quickly reattach the film to the top of the plate in the exact same position as before. Let the solution incubate for 1 hour at room temperature.
8. Carefully open the plastic film in a fume hood and transfer each paper disc to a scintillation vial containing 4 ml scintillation fluid and close the vials. Use small tweezers to manipulate the discs.
9. Centrifuge the remaining acid solution at maximum speed (≥ 14,000 × g) for 10 min at 4 °C
10. Transfer 400 μL of the supernatant to a scintillation vial containing 4 ml scintillation fluid.
11. Also add 500 ul of FAO medium to a scintillation vial containing 4 ml for supernatant radioactivity count.
12. Measure radioactivity in the filter paper, which corresponds to CO2 produced from fatty acid oxidation, using a beta counter. Radioactivity in acid fraction corresponds to acid-soluble metabolites from fatty acid metabolism (Acetyl-CoA, acyl-carnitines, etc).
13. Follow all the disposal protocols described in your risk assessment.