Simultaneous Determination of Intracellular Reactive Oxygen Species and Apoptosis Signal

A novel method for simultaneous determintation of intracellular reactive oxygen species and apoptosis signal was developed by microchip capillary electrophoresis with laser induced fluorescence detection.Alexa Fluor 488 annexin V was used to label phosphatidyl serine in outer surface of the apoptosis cells,and dihydrorhodamine 123(DHR123) was used to convert ROS to the fluorescent rhodamine 123(Rh123) intracellularly.The cells were diluted with PBS to a final density of 1.2×106 cells/mL,and then crushed by a repetitive freeze thaw method.The supernatant was separated in 1 min using 20 mmol/L borate buffer(pH 9.2) as buffer medium,with a separation voltage of 1.2 kV and an injection time of 60 s.Under the optimized experimental conditions,the calibration of Rh123 was linear in the range of 0.5-3 μmol/L with a correlation coefficient(r) of 0.998.The limit of detection(S/N=3) was 0.058 μmol/L.The established method was applied in the HepG2 cancer cell,and ROS in cells was quantified as 0.16 μmol/L before and 1.77 μmol /L after apoptosis.