Incucyte flr imaging system software

Nuclight Red cell lines were seeded at 20,000 cells in a 96-well plate (SYO-WT, SYO-OE, Fuji WT, Fuji OE, HTB-93-WT, and HTB-93-KDs) 1 day before assay. Cells were treated with either 25 μmol/L G6PDi-1 (72 hours), 2 mmol/L BSO (24 hours), 1.2 μmol/L D9 (12 hours), 250 μmol/L deferoxamine (5 hours), erastin (dosage indicated per cell line 48 hours), or 1 μmol/L ACXT-3102 (5 hours). For rescue of erastin treatment with lipophilic antioxidants or ferrostatin, the dosages were as follows: 200 μmol/L Trolox, 100 μmol/L a-tocopherol, and 10 μmol/L ferrostatin. Erastin rescue experiments were treated for 72 hours. Phenol red-free media containing drug concentration as well as 50 nmol/L of YOYO-1 iodide (Y3601, Thermo Fisher Scientific Inc) cell death marker replaced growth media. Cell death was measured via the YOYO-1 iodide probe, and live-cell analysis was measured using Nuclight Red (4717, Sartorius). Cell death was analyzed using IncuCyte FLR imaging system software (Sartorius). For quantification of death, YOYO-1 florescence was normalized to total counts for each respective well. The drugs utilized in these experiments were obtained as follows: BSO (14484, Cayman Chemical), D9 (5921, Tocris), deferoxamine (S5742, Selleckchem), Trolox (93510, Millipore-Sigma), a-tocopherol (1667600, Millipore-Sigma), and ferrostatin (S7243, Selleckchem).