Dapi pbs

Myotubes were differentiated for 8 d in 24-well culture plates in the presence of rIL-15 (0, 1, 25, 100 ng/mL), rTNFα (0, 1 ng/mL) or both cytokines (rIL-15 25 ng/mL and rTNFα 1 ng/mL). Media were renewed every 2 d. The culture medium was removed and the cells fixed with 2% formaldehyde in PBS for 30 min. Following permeabilization in 100% methanol for 10 min, wells were blocked with 5% goat serum in PBS for 30 min. Primary antibodies were diluted (Desmin, 1:1000, Dako) in 1% BSA/PBS and 150 μL was added per well for 1 hour. Wells were subsequently incubated with 150 μL/well secondary antibody (Goat anti-Mouse IgG (H + L), Alexa Fluor® 488 conjugated, Thermo Fisher) for 1 hour in the dark. Each well was washed with PBS and 150 μL/well DAPI/PBS (1:5000, Cell Signalling Technology) was added for 5 min in the dark. Wells were further washed with PBS, a drop of mountant added to each well (ProLong Diamond Antifade, Thermo Fisher) and a coverslip applied.

Myotubes were differentiated for 8 d in the presence of ACM secretome or recombinant resistin protein. The details of cytokine concentrations, as well as the timing and duration of such stimulations, are described in the relevant results section and Figure legend. Media were renewed every 2 d. The culture medium was removed and the cells fixed with 2% formaldehyde in PBS for 30 min. Following permeabilization in 100% methanol for 10 min, wells were blocked with 5% goat serum in PBS for 30 min. The primary antibody was diluted (Desmin, 1:1000, Dako) in 1% BSA/PBS and 150 μL was added per well for 1 hour. Wells were subsequently incubated with 150 μL/well secondary antibody (Goat anti-Mouse IgG (H + L), Alexa Fluor® 488 conjugated, Thermo Fisher) for 1 h in the dark. Each well was washed with PBS and 150 μL/well DAPI/PBS (1:5000, Cell Signalling Technology) was added for 5 min in the dark. Wells were further washed with PBS, a drop of mountant added to each well (ProLong Diamond Antifade, Thermo Fisher) and a coverslip applied.