We have adopted the double staining technique to establish the apoptotic potential of the blank micelles, free 2 ME, and the formulated 2 ME-loaded mixed micelles in PC-3 cell line. In brief, the PC-3 cells were exposed to the samples in a 12-well plate, where the initial density of the seeded cells was maintained at 105 cells per well (Zhang et al., 2020 (link)). IC50 concentration of 2 ME was maintained in the wells, which were incubated at 37°C for 24 h. Thereafter, the cells were washed with phosphate buffer in order to remove the micelles and free drug from the wells, and the apoptotic potential of the agents was measured using the conjugate of Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide following the instruction provided with the apoptotic detection kit (BD Bioscience, United States). Finally, the flow cytometer (FACS Calibur, BD Bioscience) was used to analyze the cells, and the obtained data were analyzed using the Multicycle software (Phoenix Flow Systems, San Diego, CA).
Apoptotic detection kit
Manufactured by BD
Sourced in United States
The Apoptotic Detection Kit is a laboratory-grade product designed to detect and analyze apoptosis, a programmed cell death process. The kit provides the necessary reagents and protocols to perform apoptosis assays, enabling researchers to study this fundamental biological phenomenon.
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4 protocols using apoptotic detection kit
1
Apoptotic Potential of 2-Methoxyestradiol Micelles in PC-3 Cells
2
Cell Cycle and Apoptosis Analysis
3
Annexin V-FITC Detection of Apoptosis
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To analyze apoptosis, cells were digested with trypsin and washed with PBS. Apoptotic detection kit was from BD (BD Biosciences). BD Pharmingen-FITC Annexin V Apoptosis Detection kit cat. no. 556547) was used and staining was conducted according to the kit instructions. The cells was analyzed by BD FACSCalibur flow cytometry facility. Annexin V-FITC+/PI− represented the apoptotic cell population.
4
Apoptosis Evaluation of 2ME-Loaded Nanoparticles
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We also implemented a double staining strategy to evaluate the apoptosis-inducing potential of the blank micelle, the free 2 ME and 2ME-ALA NPs in the MCF-7 cell line. In brief, MCF-7 cells were exposed to samples in a 12-well plate, where the initial density of the seed cells was preserved at 105 cells per well. (Zhang et al, 2020 (link)). A 10 μM concentration of 2 ME was maintained in the wells, which were incubated at 37°C for 24 h. Thereafter, the cells were washed with phosphate buffer in order to remove the micelles and free drug from the wells, and the apoptotic potential of the agents was measured using the conjugate of Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide following the instruction provided with the apoptotic detection kit (BD Bioscience, USA). Finally, the flow cytometer (FACS Calibur, BD Bioscience) was used to analyze the cells, and the obtained data were analyzed using the Multicycle software (Phoenix Flow Systems, San Diego, CA).
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