Caspase 3 7 activity

Grown BEC-organoids were treated with the FA mix for 4 days, and triglyceride levels were measured with a Triglyceride kit (Abcam, ab65336) following the manufacturer’s instructions. Cell-titer Glo (Promega, G7570) was used to investigate cell viability. For functional assays involving single BECs, grown organoids were dissociated into single cells. 10,000 BECs were seeded, and organoid formation was allowed for 7 days. Cell viability, apoptosis, and cell death were investigated using Cell-titer Glo, Caspase 3/7 activity (Promega, G8091), and Nucgreen Dead 488 staining (Invitrogen, R37109), respectively, according to the protocol of manufacturers. For cell death staining, organoids were imaged using ECLIPSE Ts2 inverted microscope (Nikon).

For cell apoptosis assay, 20 × 10⁵ cells per well in the presence or absence of recombinant EDIL3 treatment were cultured under serum-deprivation in 6-well plates. Adherent cells were detached with 0.25% trypsin without EDTA in 1 × PBS. Cells were harvested in complete RPMI 1640 or DMEM medium and centrifuged at 1000 rpm for 5 min. Each of the cells were washed with 1 × PBS, stained with 50 μg/ml propidium iodide (PI) and Annexin V-FITC (BD Pharmingen, USA) following the manufacturer's instructions. The percentage of Annexin V (+) and PI (−) cells were analyzed by flow cytometry. For anoikis assay, Annexin V/PI staining and Caspase-3/7 activity (Promega, USA) were performed as previously described [15 (link)].