A detection kit containing Annexin V-fluorescein isothiocyanate and propidium iodide (PI), which determines translocation of phosphatidylserine to the cell surface, was utilized to quantify the amount of apoptotic cells. First, 5×104 HepG2 cells were allowed to adhere in six-well plates. After exposure to Ccn suspension, Ccn-BNPs, or Ccn-BNP-GA for 24 hours, the apoptotic and live cells were harvested by centrifugation (at 1,000 rpm for 5 minutes). Cells in the sediment were then resuspended in 1.5 mL of PBS and stained with Annexin V-fluorescein isothiocyanate and PI according to the manufacturer’s instructions. The number of apoptotic cells was monitored by measuring the fluorescence of the cells with an FCM instrument (BD FACS Vantage SE). Live, early apoptotic, late apoptotic, and necrotic cells are designated as Z1, Z2, Z3, and Z4, respectively.
For analysis of the cell cycle, the drug-treated cells were washed, collected by centrifugation (1,000 rpm, 5 minutes), fixed in chilled 70% ethanol, and labeled with PI and RNase. The samples were analyzed by FCM (BD FACS Vantage SE).
A competitive binding experiment was also performed, which involved preincubation of 135.73 μmol/L free GA for 2 hours, with addition of Ccn-BNP-GA to each well at a predetermined time point. The subsequent procedure was the same as that as described in the assessment of apoptosis and cell cycle.
For analysis of the cell cycle, the drug-treated cells were washed, collected by centrifugation (1,000 rpm, 5 minutes), fixed in chilled 70% ethanol, and labeled with PI and RNase. The samples were analyzed by FCM (BD FACS Vantage SE).
A competitive binding experiment was also performed, which involved preincubation of 135.73 μmol/L free GA for 2 hours, with addition of Ccn-BNP-GA to each well at a predetermined time point. The subsequent procedure was the same as that as described in the assessment of apoptosis and cell cycle.