Cflow sampler v 1

To monitor the generation of intracellular ROS, viable HSC-3 cells were pre-treated with NAC for 1 h at 37°C, followed by 10 and 20 µM of LH for 24 h. The generation of intracellular ROS was detected via flow cytometry with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the peroxide-sensitive fluorescent probe, which may be converted to DCFH and then oxidized to the fluorescent compound DCF in the presence of ROS in cells. The cells treated with NAC and LH were harvested, washed with PBS, mixed with 10 µM DCFH-DA and incubated at 25°C for 30 min in the dark. The cell suspension was subjected to the flow cytometer and the fluorescence signal was detected for intracellular ROS measurement via flow cytometry (BD FACS Accuri C6; BD Biosciences) and data analysis of intracellular ROS was performed using a CFlow Sampler v.1.0 software (BD Biosciences).

Viable HSC-3 cells treated with different concentrations of LH (with or without pre-treatment of 5 mM NAC for 1 h) for 24 h were harvested and washed with ice-cold PBS. Cells were dual-stained with Annexin V-FITC and PI at 25°C for 20 min, followed by FCM analysis according to the manufacturer's protocol of the Annexin V-FITC/PI apoptosis detection kit. Data acquisition and analysis of the cell apoptosis were performed using a flow cytometer (BD FACS Accuri C6; BD Biosciences) and CFlow Sampler v.1.0 software (BD Biosciences).

The changes in MMP were detected via staining cells with JC-1, a fluorescent probe for MMP detection. HSC-3 cells treated with LH (with or without pre-treatment of 5 mM NAC for 1 h) for 24 h were harvested, washed with ice-cold PBS and stained with 5 mg/ml JC-1 at 37°C for 30 min in the dark. Data acquisition and analysis of MMP were performed by flow cytometry (BD FACS Accuri C6; BD Biosciences) and CFlow Sampler v.1.0 software (BD Biosciences).