Cells were counterstained with Hoechst 333342, by supplementing 1 μM Hoechst 333342 into the growth medium for 5 min before the initial wash with HBSS, after carboxy-H2DCFDA treatment.
× 15 cm × 2 μm) over a 25-min linear gradient from 4%to50%0% buffer B (80% acetonitrile and 0.08% formic acid in Milli-Q water (v/v)), followed by another 2 min to 90% buffer B. The column was then washed by holding the 90% buffer B for 10 min before returning to initial conditions of 96% buffer A (0.1% formic acid in Milli-Q water (v/v)).
These conditions are held for 15 min before returning to the initial conditions.
The solvent gradient was as follows: 0 min, 50%% B; 50 min, 75%% B; 51 min, 98 % B, 55 min, 98 % B, and then held for 10 min before returning to the initial conditions.
At 21 min, the flow was lowered to 0.2 mL/min and 100% B was used to wash the column for 10 min before returning to the initial conditions.
The initial gradient was 70% B and kept for 2 min; increased to 75% B in 3 min; increased to 80% in 5 min; increased to 90% in 3 min; increased to 99% in 3 min and kept at 99% for 5 min before switching back to initial condition.
For chromatographic separation, a Hydrosphere C18, 2.1 × 33 mm, 3 μm column (YMC Co. Ltd ,Kyoto, Japan) was used and the content of mobile phase B was increased from 2.0%to25.0%0% over 3.3 min, from 25.0% to 30.0% over 2 min, from 30.0% to 65.0% over 2.5 min and further increased from 65.0% to 95.0% over 0.1 min. The composition was held for 0.9 min before reconditioning at the initial conditions.
The column was allowed to re-equilibrate for 3 min under initial conditions before subsequent sample injections.
The gradient elution was used with the following conditions: 5% eluent B maintained for 3 min, followed by a linear increase to 45% of eluent B at 25 min, then a further increase to 90% at 30 min, conditions were held constant for 2 min before being decreased to the initial conditions at 34 min followed by a 6 min isocratic wash at 5% to re-equilibrate the column.
The initial oven temperature was held at 50°C for 2 min and was programmed to increase at 20°C per min to 325°C and held for another 11 min, before cycling back to the initial conditions.
