Epics xl mcl flow cytometry

The apoptotic status of PBMECs was evaluated by measuring the exposure of phosphatidylserine on the cell membranes using annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining [50]. The BD Pharmingen annexin V–FITC Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) was used for the apoptosis assay. PBMECs (2 × 10⁴ cells/well) were plated in a 12-well plate, and after 24-h incubation, the cells were treated with graded concentrations of Met (0, 1, 2, 3, 5, and 10 mM) for 12 h and harvested. The cell pellets were washed twice after centrifugation with cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, pH 7.4) and suspended in 100 μL of 1 × binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4). The cells were incubated with 5 μL annexin V–FITC and 10 μL PI at room temperature for 15 min in the dark. After the incubation, 400 μL of 1 × binding buffer was added to each tube. The cells were analyzed immediately by Epics XL-MCL Flow Cytometry (Beckman–Coulter, Cassina de Pecchi, Italy).

The apoptotic status of HT-29 cells was evaluated by measuring the exposure of phosphatidylserine on the cell membranes using Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) staining [50 (link)]. The BD Pharmingen Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Frankrin Lakes, NJ, USA) was used for the apoptosis assay. HT-29 cells were plated in a 24-well plate (1 × 10⁶ cells mL⁻¹), and after a 24 h incubation, the cells were treated with graded concentrations of metformin (0, 10, 25, and 50 mmol/L) for 24 and 48 h, and harvested. After centrifugation, the cell pellets were washed twice with cold phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, pH 7.4) and suspended in 100 µL of 1× binding buffer (10 mM Hepes/NaOH, 140 mM NaCl, 2.5 mM CaC_l2, pH 7.4). The cells were incubated with 5 µL Annexin V-FITC and 10 µL PI at room temperature for 15 min in the dark. After the incubation, 400 µL of 1× binding buffer was added to each tube. The cells were analyzed immediately by Epics XL-MCL Flow Cytometry (Beckman Coulter, Cassina de Pecchi, Italy).