Annexin 5 pi kit

Cell viability was assessed using an Annexin V/PI kit (Biolegend). Annexin V and PI were added to the cell samples post-transfection at 24, 48, and 72 h according the manufacturer’s recommendation, incubated for 15 min at room temperature in the dark, and followed by immediate analysis by flow cytometry (FC500 flow cytometer, Beckman Coulter). Data was processed with Kaluza® (Beckman Coulter) flow analysis software.

The effect of EC359 on cell viability of EC cells was assessed by using MTT cell viability assay as previously described [25 (link)]. To test the effect of EC359 on the viability of CSCs, CellTiter-Glo assays were performed (Promega, Madison, WI, USA). For clonogenic survival assays, EC cells were seeded in triplicate in 6 well plates (500 cells/well), after overnight incubation cells were treated with vehicle or EC359 for 5 days and after 2 weeks, colonies that contained ≥50 cells were counted and used in the analysis. The effect of EC359 on apoptosis was analyzed by using the Annexin V/PI kit as per the manufacturer’s instructions (BioLegend, San Diego, CA, USA). Briefly, EC cells were treated with either vehicle or EC359 for 48 h and cells were harvested at a density of 1 × 10⁶ cells/mL in Annexin V binding buffer. Following this step, 100 µL of cell suspension was incubated with Annexin V FITC and propidium iodide (PI) for 15 min at room temperature in the dark. Lastly, 400 µL of Annexin V binding buffer was added to each sample and stained cells were analyzed using flow cytometry.

HepG2 cells were exposed to the high glucose culture medium for 2 h. Then, treatments such as metformin alone, exosome alone, and the combination of metformin and exosome are added to the HepG2 cells culture medium. BioLegend AnnexinV-PI kit was used to evaluate the percentage of apoptosis and necrosis in different treated groups (BioLegend, USA). At the end of the treatment and incubation period, the cells were removed from the bottom of the flask with 0.25% trypsin-EDTA (Sigma, USA). After neutralizing trypsin with 10% FBS containing DMEM medium, the cell suspension was centrifuged for 5 min at 1500 rpm. Then the supernatant was removed, and the cell pellet was dissolved in 500 μl binding buffer and centrifuged at 1500 rpm for 5 min. Then in the dark environment, 5 μl AnnexinV dye was added to each microtube and incubated for 20 min in the dark. Then, 5 μl of PI dye was added to the cell suspension and analyzed by FACSCanto II flow cytometry within 1 h. Flow cytometric data were also analyzed by Flowjo software. This study was performed independently 5 times on different groups of HepG2 cells in triplicates.