Mantra imaging platform

After treatment, worms were washed off from the plates and centrifuged at 600×g for 1 min. The worms were washed thrice and fixed with 40% isopropanol for 3 min, centrifuged at 600×g for 1 min, and stained with Nile red as per the protocol of Escorcia et al. (38 (link)). The images were acquired using a FITC filter on an Olympus BX43 microscope equipped with a Mantra imaging platform (PerkinElmer, USA) and further processed on Inform 2.2 software suite (PerkinElmer, USA).

The HepG2 spheroids (n = 3 per group), after treatment with Livogrit (0, 30 µg/mL) and pioglitazone (10 µM), were transferred at a density of one spheroid/ chamber of Kline concavity slide (HiMedia, Mumbai, India). The spheroids were washed twice with HBSS and a staining solution comprised of CellROX green solution (5 µM) and LipidSpot lipid droplet stain (1:1000) in DMEM was added on each concavity. The spheroids were kept for incubation of 45 min at 37°C. Upon incubation, spheroids were washed with HBSS and further stained with Hoechst 33342 (1:1000). For evaluation of lipid accumulation in rat primary hepatocytes treated with Livogrit (0, 30 µg/mL) and pioglitazone (10 µM), cells were washed with HBSS and stained by LipidSpot (1:1000) for 20 min. The cells were fixed via 4% formaldehyde and mounted with ProLong Diamond antifade mountant with DAPI (Invitrogen, Waltham, MA, USA). Microscopy was performed by Olympus BX43 microscope equipped with Mantra imaging platform (Perkin Elmer, Waltham, MA, USA) and further processed on Inform 2.2 software suite (Perkin Elmer, Waltham, MA, USA).