6 well multidish

The ability of C2C12 cells, GRMD MABs and SKM cells to differentiate into skeletal muscle was assessed in vitro by exposing the cells upon reaching 80% of confluence to a specific differentiation medium composed of DMEM supplemented with 2% horse serum (Euroclone), 1% P/S, 1% L-glutamine for 4 to 10 days (53 ). C2C12 cells were induced to differentiate on a 6-well multidish (Nunc), while GRMD MABs and SKM cells were seeded onto matrigel (BD Biosciences) coated dishes. When cells were transduced with the tamoxifen-inducible MyoD-ER lentiviral vector, 1 μM 4-hydroxy-tamoxifen (Sigma-Aldrich) was added in the growth medium that was replaced 24 h later by differentiation medium supplemented with 1 μM 4-hydroxy-tamoxifen. Half of the medium was replaced with fresh differentiation medium every other day. Immunofluorescence (IF) staining for myosin heavy chain (MyHC) was performed to confirm multinucleated myotubes (see specific section for ‘IF staining’). Differentiation of GRMD MABs in smooth muscle-like cells was induced by treating the cells for 10 days with differentiation medium supplemented with 5 ng/ml transforming growth factor β1 (Sigma-Aldrich) (62 (link)). Smooth muscle differentiation was confirmed by IF staining for α-smooth muscle actin (αSMA; see specific section for ‘IF staining’).

Cells were electroporated with the Amaxa Nucleofector II (Lonza). For C2C12 myoblasts, the Cell Line Nucleofector Kit V and the program B-32 were used. Whilst for GRMD MABs, the Human MSC Nucleofector Kit and the program U-23 were applied. Transfections were optimized to obtain good efficiency with low levels of toxicity, modifying the amount of plasmids used and the transposase: transposon ratio. Briefly, cells were trypsinized, washed in PBS (Life Technologies) and counted; 1×10⁶ cells were electroporated and subsequently seeded in a single dish of a 6-well multidish (Nunc). The day after, the medium was replaced with fresh medium. Transfection efficiency was assessed by calculating the percentage of GFP+ cells at 24, 48 and 72 h post-electroporation (EP) by fluorescence microscopy and/or fluorescence activated cells sorter (FACSCanto™ flow cytometer, Becton Dickinson). In this case, transfected C2C12 cells or GRMD MABs were therefore trypsinized, washed in PBS, counted and resuspended into PBS supplemented with 1% FBS and 2mM EDTA (Life Technologies). Analyses were performed with FACSDiva software. Where indicated, GFP positive C2C12 cells or GRMD MABs were enriched respectively at day 4 and 7 days post-EP by FACS sorting (BD FACSAria, Becton Dickinson). Transfected cells were monitored for 28–30 days post-EP to assess transposition efficiency.