H-2Kb-tsA58 mdx myoblasts (H2K mdx cells) were cultured and differentiated as described previously (22 (link)32 (link, link, link, link, link, no link found, no link found, no link found, link, no link found)). Briefly, 60–80% confluent, myoblast cultures were treated with trypsin (Thermo Fisher Scientific, Scoresby, VIC, Australia) and seeded on 24-well plates pretreated with 50 μg/mL poly-D-lysine (Merck Millipore, Bayswater, VIC, Australia), followed by 100 μg/mL Matrigel (Corning, supplied through In Vitro Technologies, Noble Park North, VIC, Australia) at a density of 2.5 × 104 cells/well. Cells were differentiated into myotubes in low-glucose Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) containing 5% horse serum (Thermo Fisher Scientific) by incubating at 37 °C, 5% CO2 for 24 h.
ASOs were complexed with lipofectin transfection reagent (Thermo Fisher Scientific) at the ratio of 2:1 (w:w) (lipofectin:ASO) and used in a final transfection volume of 500 μL/well in a 24-well plate as per the manufacturer's instructions, except that the solution was not removed after 3 h. For “naked” transfection, the ASOs were mixed directly with Optimem reduced serum medium and added to the cells. For long-term incubations, the cells were collected at 1-, 3-, and 5-d intervals. All experiments were repeated independently at least three times unless specified.
ASOs were complexed with lipofectin transfection reagent (Thermo Fisher Scientific) at the ratio of 2:1 (w:w) (lipofectin:ASO) and used in a final transfection volume of 500 μL/well in a 24-well plate as per the manufacturer's instructions, except that the solution was not removed after 3 h. For “naked” transfection, the ASOs were mixed directly with Optimem reduced serum medium and added to the cells. For long-term incubations, the cells were collected at 1-, 3-, and 5-d intervals. All experiments were repeated independently at least three times unless specified.
