8 hepe

Murine 3T3-L1 preadipocytes were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1% penicillin-streptomycin and 10% fetal bovine serum in a 5% CO₂ incubator at 37°C. To induce adipogenesis, 3T3-L1 cells were plated in 24-well plates and grown to confluence. Cells were differentiated in cell culture medium containing 0.5 mM 1-methyl-3-isobutylxanthine, 0.25 μM dexamethasone, and 10 ng/mL insulin that was supplemented with vehicle control or 5μM 8-HEPE (Cayman Chemicals), arachidonic acid (AA) (Nu-Chek Prep), or EPA (Nu-Chek Prep) for 3 days. AA, EPA or 8-HEPE were administered from ethanol stock solutions. Medium was subsequently changed to DMEM containing 10 ng/mL insulin and 5μM 8-HEPE, AA, or EPA and replaced every two days with freshly respective medium. After adipocyte differentiation, cells were washed with phosphate-buffered saline, fixed with 10% formalin and stained with Oil Red O (EMB Bioscience) solution following the manufacturer’s protocol. Fixed plated cells were then washed 3 times with deionized water, then images were taken with an EVOS M7000 microscope (Thermo Fisher Scientific). For quantification of Oil Red O, cells were harvested with isopropanol, and absorbance was determined at 540nm. Amount of absorbed Oil Red O is directly proportional to cellular lipid content.