Flow cytometry assays were processed using monoclonal antibody Ki-67 and Annexin V/propidium iodide (PI) double staining, to evaluate the degree of proliferation apoptosis, as in our previous study [23]. Briefly, SH-SY5Y cells were seeded into 6-well plates (Nest Biotech., China) at a density of 1 × 105 cells per well. After pretreatment with different concentrations (0.37, 1.1, and 3.3 μg/mL) of Triphala for 24 h, the cells were treated with 400 μmol/L H2O2 for an additional 20 h. Then, the cells were harvested by trypsinization, incubated with −20°C in absolute ethanol, washed, and resuspended in cell staining buffer. Subsequently, cells were fixed and the nuclear membrane was permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience Inc., San Diego, CA) before staining with anti-Ki67 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 1 h. To detect the apoptosis, cells were incubated using the FITC Annexin V Apoptosis Detection Kit with PI (Dojindo Molecular Technologies Inc.), at room temperature for 15 min. Afterwards, the fluorescent staining was detected and analyzed using a flow cytometer (BD Biosciences, San Jose, CA). The mean fluorescent intensity for Ki-67 was calculated using FlowJo VX software (Tree Star Inc., Ashland, OR).

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