Myotube-to-macrophage conditioned medium

Human primary myotube cell culture

• Muscle cell source: Primary muscle cells were isolated from vastus lateralis biopsies derived from men (Age: 50±10 years; BMI: 22.9±2.4 kg/m²) with or without a clinically diagnosis of type 2 diabetes. The ethical committee at Karolinska Institutet approved protocols and informed consent was obtained from the volunteers. Cells are grown in incubators maintained at 37 degrees with a humidified atmosphere of 7.5 % CO2. The absence of mycoplasma contamination is routinely confirmed by PCR.
• Proliferation medium: F12/DMEM, 25 mM glucose; (#31331, Thermo Fisher Scientific) supplemented with 100 units/mL penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of Amphotericin B and 20% fetal bovine serum.
• Differentiation medium: DMEM with GlutaMAX (#31966, Gibco) containing 20 % Medium 199 (#31150, Gibco), HEPES buffer (0.02 M; #15630, Gibco), zinc sulfate (0.03 μg/mL), vitamin B₁₂ (1.4 μg/mL; Sigma-Aldrich), insulin (10 μg/mL; Actrapid; Novo Nordisk), and apo-transferrin (100 μg/mL; #T100-5, BBI Solutions).
• Post-fusion medium: DMEM with GlutaMAX (#31966, Gibco) containing 20% Medium 199 (#31150, Gibco), HEPES buffer (0.02 M; #15630, Gibco), zinc sulfate (0.03 μg/mL), vitamin B₁₂ (1.4 μg/mL; Sigma-Aldrich) 0.5 % fetal bovine serum.
• Protocol for differentiation:
  • Seed cells in 6-well plates, grow until confluence in proliferation medium.
  • When the cells are confluent, switch to differentiation medium.
  • After 4 days, switch to post-fusion medium.
  • The formation of myotubes is monitored under the microscope. Cells are used for experiments 6-10 days after the initiation of differentiation.

Electrical Pulse Stimulation (EPS) and conditioned medium

This protocol uses the C-Pace EP Culture Pacer (IonOptix, MA). 

• Grow and differentiate primary human muscle cells in 6-well plates as described above.
• Wash cells with PBS.
• Add fresh post-fusion medium, at least 2 ml per well.
• Place the lids with carbon electrodes on the cells.
• Pulse the cells at 40 V, 1 Hz, 2 ms pulse duration for 3 h. Control cells are incubated in the presence of the carbon electrodes without electrical stimulation.
• Immediately at the end of the electrical stimulation, collect cell supernatant and transfer it in clean, sterile tubes on ice.
• Filter medium through 0.2 µm filters to remove cellular debris.
• Aliquot and freeze at -80 degrees.

The supernatant collected this way contains molecules released by skeletal muscle cells during EPS and is thereafter referred to as “conditioned medium”.

THP1 macrophage culture

THP1 human monocytes purchased from the ATCC are grown in RPMI 1640 containing 5 % fetal bovine serum and supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B. Cells are grown in incubators maintained at 37 degrees with a humidified atmosphere of 5% CO2. The absence of mycoplasma contamination is routinely confirmed by PCR. Protocol for differentiation and treatment of THP1 macrophages:

• Dilute monocytes to 2.10⁶ cells/mL in medium containing 100 ng/mL phorbol 12-myristate 13-acetate (PMA).
• Immediately add 1 ml of cell suspension per well of a 12-well plate.
• After 24h, check differentiation under the microscope: the cells should now be adhering to the plates.
• Wash twice with PBS to remove PMA and add 1 ml of fresh growth medium.
• Allow macrophages to stabilize for 24h.
• Replace the medium with the post-fusion medium used for myotubes and incubate for 24h.
• Add 1 ml of conditioned medium per well and incubate for 3 h, 6 h, or 24 h.
• Extract RNA with the E.Z.N.A.® Total RNA Kit I (Omega Bio-tek).