Fitc annexin 5 apoptosis detection kit 2

For semi-quantitative analysis, apoptotic cells were detected by the EB/AO solution kit (Merck Millipore, Darmstadt, Germany), at a cell density at 2.5 × 10⁴ in 96 well-plates, and were treated with at SC and 2-fold SC (2xSC) of Androg for 48 h., according to the manufacturer’s protocol, and observed under an Eclipse NiU (confocal) microscope (Nikon Corporation, Tokyo, Japan). The apoptotic cells were randomly counted from three fields in each treatment condition. For the quantitative determination of the Androg-induced apoptosis, a FITC Annexin V Apoptosis Detection Kit II (Invitrogen, Carlsbad, CA, USA) was used. Briefly, a cell density of 1.5 × 10⁵ was seeded onto a 24 well-plate and treated with SC and 2xSC Androg for 48 h., and then, cells were stained with annexin V-FITC and propidium iodide (PI) for 15 min at room temperature (RT) according to the manufacturer’s protocol. Apoptotic cells were then assessed using a BD FACSCanto™ II Cell Analyzer (BD bioscience, Franklin Lakes, NJ, USA). Quantities of 50 µg/mL of Cycloheximide (CHX) and DMSO were used as the chemical-induced apoptosis and blank controls.

The quantitative analysis of apoptotic cell death was performed using the FITC Annexin V Apoptosis Detection Kit II (Invitrogen). HepG2 cells were transfected with siRNAs. After 24 h, the cells were plated at 1×10⁶ cells/well in a new 6-well plate, incubated under normoxia or hypoxia for a further 24 h, and the cells were analyzed by fluorescence-activated cell sorting (FACS) using an ARIA3 apparatus (BD Biosciences, San Jose, CA, USA).