Dulbecco s phosphate buffer saline dpbs

All mammalian cell lines were purchased from ATCC, and cultured in RPMI1640 medium (HiMedia) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) (Invitrogen) and 1x penicillin-streptomycin (MP Biomedicals) at 37 °C with 5% (v/v) CO₂. All cell lines were routinely stained with DAPI to ensure they were devoid of any mycoplasma contamination. All cell viability studies for MGR1 and MGR2 treatments were done using a TC20 Automated Cell Counter with Trypan Blue reagent (Bio-Rad) as per manufacturers instructions. Briefly, cells were treated with varying concentrations (0 – 40 μM) of MGR1 or MGR2 for 4 h, at which point, the cells were detached by trypsinization and live cells were estimated as per manufacturers protocol. For lipid measurements, 2x10⁶ cells were washed with sterile Dulbecco’s Phosphate Buffer Saline (DPBS) (HiMedia) (3X) and treated with MGR1 (2 μM) or MGR2 (2 μM) or DMSO for 4 h at 37 °C and 5% (v/v) CO₂ in 5 mL of aforementioned media (10 cm tissue culture dish). Post treatment, the cells were washed again with sterile DPBS (3X), and the lipids were extracted immediately using the protocol described below. All cellular ROS chemifluorescence imaging were performed using the DCF dye (10 μM, 10 min, Thermofisher Scientific) on an EVOS® FL Auto Imaging System (Life Technologies, Thermofisher Scientific) as per manufacturer’s instructions.