Facstar flow cytometer

A lentivirus vector encoding an shRNA targeting OIP5 (shO-1 target sequence: 5'-CCGGGCATCAGAGATGGATATTCAACTCGAGTTGAATATCCATCTCTGATGCTTTTTG-3' and shO-2 target sequence: 5'-CCGGCCATGTGTCCTCTGATCTAAACTCGAGTTTAGATCAGAGGACACATGGTTTTTG-3'), or an shRNA non-target control (NT sequence: 5'-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTT-3') were used for transduction of Huh7 and HLK2 cells according to the manufacturer's instructions (Sigma-Aldrich, St Louis, MO, USA). In brief, 5 × 10⁵ cells were incubated overnight in a 6-cm plate and transduced with lentiviral particles at a multiplicity of infection (MOI) of 1 in the presence of 8 μg/ml of polybrene. Western blot analysis was performed to confirm the knockdown of OIP5 expression. For FACS analysis, cells (2.5 × 10⁵) were cultured in a six-well plate, transduced with lentiviruses for 18 h, and treated with fresh medium. At 48 h post-transduction, cells were harvested, fixed with 70% cold ethanol, and stained with PI buffer (10 μg propidium iodide and 1 mg/ml RNAse in 1.1% sodium citrate) at 37°C for 30 min. The percentage of cells in each phase of the cell cycle was determined with a FACStar flow cytometer (Becton Dickinson, San Jose, CA, USA).

Adenovirus-infected HPASMCs were maintained in serum-free M231 medium for 24 hours and then stimulated with 10 ng/mL PDGF-BB for different periods. The cells were trypsinized, fixed in 70% ethanol at 4°C overnight, washed twice with ice-cold PBS, and incubated with RNase and propidium iodide. The cell-cycle phase was analyzed by flow cytometry using a Becton Dickinson FACStar flow cytometer and the Becton Dickinson CellFIT software.

Apoptosis was determined by staining cells with annexin V conjugated to green-fluorescent fluorescein isothiocyante (annexin V-FITC) and propidium iodide (PI) labeling29 (link). Briefly, 1 × 10⁵cells/mL were incubated with JB for 24 h. Afterwards, the cells were washed twice with ice-cold PBS, and then 5 μL of annexin V-FITC (PharMingen, San Diego, CA, USA) and 5 μL of PI (1 mg/mL) were applied to stain cells. The status of cell staining was analyzed by using the FACStar flowcytometer (Becton Dickinson, New Jersey, USA). Viable cells were negative for both PI and annexin V-FITC; apoptotic cells were positive for annexin V-FITC and negative for PI, whereas late apoptotic dead cells displayed strong annexin V-FITC and PI labeling. Nonviable cells, which underwent necrosis, were positive for PI, but negative for annexin V-FITC.

Cellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA; Ex/Em = 522 nm/595 nm; Invitrogen Molecular Probes) as previously described^{6 (link),37 (link)}. In brief, 1 × 10⁶ cells in 60-mm culture dishes (BD Falcon) were pretreated with 2 mM NAC or 10 μM BSO for 1 h and then treated with PG at an indicated concentration (100–1600 μM) for 24 h or other indicated time points. Cells were then washed with PBS and incubated with 5 µM CMFDA at 37 °C for 30 min. CMF fluorescence intensity was determined using a FAC Star flow cytometer (Becton Dickinson). CMF-negative (−) staining cells indicate depletion of GSH in cells. The mean CMF levels in cells, except for CMF-negative (GSH-depleted) cells, were expressed as a percentage of those of control cells.

Apoptosis was determined by staining the cells with FITC Annexin V Apoptosis Detection Kit II (Becton-Dickinson Biosciences, CA, USA). The cells were harvested after incubation with UA (0, 10, 25 or 50 µM) 48 h and stained with Annexin V-FITC for 30 min. at 37 °C. The cells analyzed using a FACStar flow cytometer (Becton-Dickinson, San Jose, CA, USA).