Yo pro 1 pi

A density of 2 × 10⁶ Huh-7 cells on 6-well plates was maintained with 25 μM curcumin for a given period of time ranging from 0 to 48 h depending on the experiments. After treatment, cells were trypsinized and then harvested, washed, and resuspended together with their supernatant in PBS. 3,3′-Dihexyloxacarbocyanineiodide [DiOC₆(3)] was added at 40 nM final concentration for ΔΨm determination, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 5 μM for peroxidase activity, and MitoSOX at 1 μM for superoxide anion. Most of the time double staining was done in order to assay simultaneously cell viability, with propidium iodide (PI, stock solution, 1 mg.mL⁻¹) for DiOC₆(3), DCFH-DA and Fluo4-AM and with TO-PRO-3 iodide (stock solution, 1 mg.mL⁻¹) for MitoSOX. A supplemental double staining was used for the distinction between viable, apoptotic, and necrotic cells with YO-PRO-1/PI (Molecular Probes) in parallel with annexin-V/PI staining done with annexin-V-FITC when needed (Immunotech, Beckman-Coulter) in the presence of calcium in order to detect the aberrant exposure of phophatidyl serine residues at the outer surface of the plasma membrane. All samples were analyzed using flow cytometry as previously described [24 (link),25 (link)] on a FACS Calibur 4C.

A density of 2 × 10⁶ Huh-7 cells on six-well plates were maintained with 25 μM of curcumin for a given period of time ranging from 0 to 48 h depending on the experiments. After treatment, cells were trypsinized, harvested, washed and then resuspended together with their supernatant in PBS. 3,3′-Dihexyloxacarbocyanineiodide [DiOC₆(3)] was added to a final concentration of 40 nM for ΔΨm determination, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) to 5 μM for H₂O₂, and MitoSOX to 1 μM for superoxide anion. Most of the time double staining was done in order to assay simultaneously cellular viability, with propidium iodide (PI: stock solution; 1 mg mL⁻¹) for DiOC₆(3), DCFH-DA and Fluo4-AM and with 2 μg/mL TO-PRO-3 iodide (stock solution; 1 mg mL⁻¹) for MitoSOX. Supplemental double staining was used for the distinction between viable, apoptotic and necrotic cells with YO-PRO-1 / PI (Molecular Probes) in parallel with annexin-V/PI staining done with annexin-V-FITC when needed (Immunotech, Beckman-Coulter). All samples were analyzed by flow cytometry as previously described^{77 (link),78 (link)} on a FACS Calibur 4C.