Corrsight spinning disk confocal microscope

Based upon the results from live cell imaging, we picked the 48‐h time point for detailed examination in fixed cells. Human primary myotubes from three different donors were grown on #1 coverslips and incubated with resveratrol for 48 h. Myotubes were fixed with 3.7% formaldehyde and stained with 286 nM DAPI (4’,6‐diamidino‐2‐phenylindole, dihydrochloride; D1306; Invitrogen) to visualize the nuclei and stained with Bodipy 493/503 (1:100) to visualize the LDs. Coverslips were mounted on glass slides with Mowiol. Imaging was performed with a FEI Corrsight spinning‐disk confocal microscope, using a 63× 1.4 N.A. oil immersion objective (Zeiss) and with a Nikon E800 fluorescence microscope (Nikon), coupled to a Nikon DS‐Fi1c colour CCD camera (Nikon), using a 40x objective. Images obtained from the Corrsight were analysed similar to the live cell images. Images from the Nikon microscope were analysed upon thresholding and Bodipy‐derived signal was corrected for the number of nuclei.

To monitor and quantify the effects of resveratrol incubation on IMCL accumulation in vitro over time, we first performed live cell imaging in myotubes from an untrained donor for 48 h with 1‐h intervals. Live cell imaging was performed using a FEI Corrsight spinning disk confocal microscope, equipped with an ORCA‐Flash 4.0 V2 CMOS camera, using a 40× 0.9 N.A. air objective (Zeiss) at 37°C. Bodipy 493/503 (D3922; Molecular probes, Fisher Scientific; 1:250) was added to the medium (and remained present throughout the imaging period) to visualize the LDs and CellMask (C10046; Invitrogen, Fisher Scientific; 1:1000) was used to visualize the plasma membranes. Upon thresholding the Bodipy‐derived signal, total lipid area, total LD number and average LD size were quantified in multiple wells.