Spectral c2 microscope

To measure the LPO level and analyze lipid droplets, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY^® 581/591 C11) (Invitrogen, Carlsbad, CA, USA) was used. This dye shifts from red to green upon oxidation. For hepatocyte isolation, 4–5 mm size samples were mechanically disaggregated using a Tissue Tearor homogenizer (Cole-Parmer, Vernon Hills, IL, USA), and purified using Ficoll (Sigma Aldrich, St. Louis, MO, USA) after centrifugation at 3000× g for 90 s. Freshly isolated hepatocytes were incubated with BODIPY 581/591 for 60 min at room temperature. Green and red fluorescence signals were captured by confocal microscopy (Nikon Spectral C2+ microscope, Tokyo, Japan). An acquisition protocol using simultaneous double wavelength excitation (laser lines Ex488/Em520 and Ex561/Em595nm, respectively) was used. Fluorescence intensity in both channels was quantified by FIJI (NIH, Bethesda, MD, USA) (National Institutes of Health, Bethesda, MD, USA).

To measure the lipoperoxidation (LPO) level and analyze lipid droplets, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY^® 581/591 C11) (Invitrogen, Carlsbad, CA, USA) was used. This dye shifts from red to green upon oxidation. Freshly isolated hepatocytes or isolated cultured skeletal fibers were incubated with BODIPY 581/591 for 60 min at room temperature. Green and red fluorescence signals were captured by confocal microscopy (Nikon Spectral C2⁺ microscope, Tokyo, Japan). An acquisition protocol using simultaneously double wavelength excitation (laser lines 488 and 568 nm) was used. Fluorescence intensity in both channels was quantified by FIJI (NIH, Bethesda, MD, USA). (National Institutes of Health, Bethesda, MD, USA).