L5900

SC-β cells were incubated with PA (Sigma-Aldrich, #P0500), SA (Sigma-Aldrich, #S4751) and LA (Sigma-Aldrich, #l5900) or solvent control (8% BSA in 0.1 M NaOH for PA and SA; ddH2O for LA) in albumin-free medium for 12–24 h^{40 (link)–42 (link),106 (link)} and dissociated into single cells by 0.25% trypsin. Cell death was measured by using an Annexin V/PI apoptosis detection kit (YEASEN, 40304ES60) and assessed by flow cytometry.

The standard media for PHH culture was supplemented with a combination of FAs in order to promote increases in the intracellular triglyceride levels stocked as LDs, as previously described [14 (link)]. This fatting media consisted of the saturated palmitic acid (P0500; Sigma-Aldrich), polyunsaturated omega-6 linoleic acid (L5900; Sigma-Aldrich) and the monounsaturated omega-9 oleic acid (O1257; Sigma-Aldrich) all at a final concentration of 0.25mM. This concentration was determined by performing cytotoxicity titration experiments prior to institution of the full experimental protocol. A supplement of 5% fatty-acid-free bovine serum albumin weight/volume (BSA) (A3803; Sigma-Aldrich) was added as a protein carrier. The media was changed daily and the steatosis induction period was 48 hours. The lean control group was incubated with standard media only throughout the experimental period.