After being cultured in either 1 or 10% FBS for 24 h, the cells were treated with a concentration gradient of recombinant CCN3 (rCCN3; Sino Biological Inc., Beijing, China; 0, 0.5, 1 and 2 µg/ml) for 24 h. Subsequently, apoptosis was determined by staining with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's protocol. To quantify apoptosis, the cells were washed with cold PBS and suspended in binding buffer. The cells were then stained with 5 µl annexin V-FITC and 5 µl PI at 4°C for 15 min, and were analyzed using FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) (22 (link)). The cells were quantified as follows: i) Annexin V negative/PI negative (viable cells); ii) annexin V positive/PI negative (cells in the initial stages of apoptosis); iii) annexin V positive/PI positive (cells in the advanced stages of apoptosis); and iv) annexin V negative/PI positive (necrotic cells).