Hcs studio 2

A multiparametric cytotoxicity assay was performed using a cellomics high content screening (HCS) reagent HitKit as per the manufacturer’s instructions (Thermo Fisher Scientific Inc., Waltham, MA, USA). This kit measures cell count, MMP, ROS and GSH.
The HepG2 cells were placed on 96 well plates in the DMEM. On the second day, the cells were treated overnight with the 6.25, 12.5, 25, 50, 100 μg/mL nano-CeO₂ and vehicle (0.1 % DMSO), respectively. After 24 h of incubation (37 °C, 5%CO₂, 100% humidity), the media were removed and the cells were stained by fluorescent probes in the same culturing medium. The fluorescent probes were: Hoechst 33342 for cell count, tetramethylrhodamine methyl ester (TMRE) for mitochondrial membrane potential, 2′,7′-dichlorofluorescein (DCFH-DA) for ROS, and finally monochlorobimane (mBCL) for glutathione. Automated live-cell multispectral image acquisition was performed on a High Content Analysis (HCA) Reader (ArrayScan XTI, Thermo Fisher Scientific Inc.). The fluorescence images were captured according to the excitation and emission wavelengths of each probe: (1) 350 and 461 nm for Hoechst 33342 on channel 1; (2) 584 and 606 nm for TMRE on channel 4; (3) 504 and 529 nm for DCFH-DA on channel 3; (4) 380 and 461 nm for mBCL on channel 2. Image analysis was performed using HCS Studio™ 2.0 software (Thermo Fisher Scientific Inc.)

The effect of SLCAs on [Ca²⁺]i release was examined quantitatively and qualitatively using Fluo 4-acetoxymethyl ester (Fluo-4 AM) dye as described previously [15 (link)]. Briefly, HeLa 1x10⁵ ml^-1 cells were treated with sophorolipids for 4, 8 and 12 h. Following incubation, the cells were washed with PBS and loaded with Fluo-4 AM (4 μM). Nuclei were stained with DAPI. An alteration in released calcium level was detected using LSCM by measuring the green fluorescence (excitation 490 nm, emission 530 nm). Data analysis was carried out using Thermo Scientific^™ HCS studio^™ 2.0 software.

The distribution of sophorolipid treated cells in cell cycle was assayed as described earlier [11 ] by DAPI staining at different time intervals. Briefly, HeLa cells were treated with sophorolipids SLCA B (16.32 μg ml^-1) and SLCA C (14.14 μg ml^-1) for time interval of 6, 12, 18 and 24 h. Following incubation, cells were stained with DAPI and the DNA content was measured using a laser-scanning confocal microscope (LSCM). Data was analysed using Thermo Scientific^™ HCS studio^™ 2.0 software to calculate the percentages of cells in G1, S and G2/M phase.