Lipid Droplet Quantification in Adipocytes

Seven days after adipogenic media and LIV treatment, cells were fixed and were stained with Oil Red O (Poly Scientific, Baywood, NY, USA, #k043), Lipid Spot 610 (Biotium, Fremont, CA, USA, #70069), and NucBlue Hoechst stain. Images were taken using a 20× objective and exported to quantify lipid droplet formation via a custom-made MATLAB program (MathWorks, Natick, MA, USA) previously published [63 (link)]. A minimum pixel intensity of 80 was used to isolate lipid droplet staining. The mean lipid droplet intensity per cell was calculated by dividing the sum of lipid droplet stain intensity by the nuclei count per image. For determining the effects of siLmna on lipid droplet formation, nuclear area, nuclear perimeter, and nuclear circularity, siCntl- and siLmna-treated cells were differentiated with previously stated adipogenic media and indomethacin (1 µg/mL) for 5 days. Cells were then stained for lamin A/C, as previously described, Lipid Spot 610, and NucBlue Hoeschst. Exported images were used to quantify lipid droplet formation, lamin A/C, nuclear area, nuclear perimeter, and nuclear circularity via the custom-made MATLAB program. A minimum pixel intensity of 80 was used to isolate lipid droplet staining and lamin A/C intensity.