Facstar cytofluorometer

Cells infected with RT and/or rAd-TRAIL were trypsinized and washed once with complete medium. An aliquots of cells (5 × 10⁵) was resuspended in 500 mL of binding buffer and stained with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI, BioVision, Palo Alto, California) according to the manufacturer’s instructions. Cell apoptosis and cell cycle were examined using FACS (FACStar cytofluorometer; BD Biosciences, San Jose, California).

Mixed cell populations containing 10% CBRH7919^tk and 90% CBRH7919^WT cells were seeded in quadruplicate in 96-well plates at 3 × 10³ cells per well. When the cells had adhered on the second day, they were exposed to resveratrol (10 µM or 20 µM), GCV (15.7 µM) and DMSO (negative control) for 24 h, and GCV (15.7 μM) was added to the culture medium in one group for 24 h more. Cell viability was assessed using the MTT assay. Absorbance values were determined at 490 nm with a microplate reader (Bio-Tek ELx800; Bio-Tek Instruments Inc., Winooski, VT, USA). To determine the rate of apoptosis, the drug-treated cells were trypsinized, fixed in 70% ethanol at 4°C and stained with 40 mg/mL propidium iodide for 30 min at 37°C. Ten thousand cells from each group were analyzed with a FACStar cytofluorometer (BD Biosciences) that was equipped with an argon-ion laser (488 nm).

SKOV-3 cells were treated with cisplatin (4 μg/mL), ZD55-MnSOD (10 multiplicity of infection [MOI]), or cisplatin (4 MOI) plus ZD55-MnSOD (4 μg/mL). After 48 hours, apoptosis cells were detected by using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining or PI staining alone following the manufacturer’s introduction. Cell apoptosis and cell cycle were examined using FACS (FACStar cytofluorometer; BD Biosciences, San Jose, CA, USA).