Cytoflex flow cytometry analyzer

Cell death was determined using the EzWay Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. HepG2 cells were grown in 6-well plates and exposed to 320 µM FTF or 0.008% DMSO (vehicle control) for 24 h with or without pretreatment with NAC (5 mM; in PBS) for 1 h (n = 3 wells per group). The medium was collected in a 15 mL tube after the end of the FTF treatment, and the cells were washed twice with PBS. Next, the cells were harvested and pelleted by centrifugation at 3000× g for 5 min at 4 °C. Subsequently, the cells were washed with 4 °CPBS and resuspended in 1× binding buffer. Next, the cells were stained with Annexin V-fluorescein isothiocyanate (1.25 µL) and propidium iodide (10 µL) and incubated in the dark for 10 min at room temperature. The stained samples were analyzed using a CytoFlex Flow Cytometry Analyzer (Beckman Coulter, Brea, CA, USA).

Cell apoptosis was determined using the EzWayAnnexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO, USA) as described previously with minor modifications [47 (link)]. The Caco-2 cells were seeded in 6-well plate and treated with fermented PM supernatants (1, 2.5, and 5%), acetate (0, 10, 20, and 30 mM), or lactate (0, 20, 30, and 40 mM) for 48 h. In addition, deionized water (5%) was used as a control. Next, the cell medium was collected in a 15 mL tube, and the cells were washed twice using PBS. Then, the cells were harvested with a 0.05% trypsin/0.53 mM EDTA solution and centrifuged at 2000× g for 5 min at 4 °C. After removing the medium supernatants, the cells were washed twice with cold serum-containing media and PBS, and then, they were resuspended in 1× binding buffer. Subsequently, the cells were added with 1.25 µL of Annexin V-fluorescein isothiocyanate and 1 µL of propidium iodide (1 mg/mL), and then, they were incubated in the dark for 30 min at 4 °C. Next, the cells were centrifuged at 1000× g for 5 min at 4 °C and re-suspended again in 1× binding buffer. The re-suspended cells were analyzed immediately using a CytoFlex Flow Cytometry Analyzer (Beckman Coulter, Brea, CA, USA).

The cells were starved with serum-free media for 24 h and treated with the optimal combination of hormones (I, 1 ng/mL; C, 5 ng/mL; P, 10 ng/mL; and E2, 0.1 ng/mL) and the optimal combination of hormones and 20% level of EAAs (Arg, 5.0 mM; His, 1.0 mM; Leu, 0.9 mM; Ile, 0.6 mM; Thr, 0.9 mM; Trp, 1.2 mM; Lys, 2.0 mM; Met, 1.2 mM; Phe, 1.5 mM; and Val, 1.5 mM) for 24 h. After treatment, the cells were detached using trypsin-EDTA solution and collected in 1.7-mL microtubes. The cells were fixed in 70% ethanol at 4°C for 16 h. Next, the cells were suspended in PBS of 500 μL supplemented with RNase A (2 mg/mL, final concertation; Sigma-Aldrich, USA) and incubated at 37°C for 1 h. Subsequently, propidium iodide (0.1 mg/mL, final concentration; Sigma-Aldrich, USA) was added to the cells in 1.7-mL microtubes for staining, and the stained cells were analyzed for cell cycle distribution using a CytoFlex Flow Cytometry Analyzer (Beckman Coulter, Brea, CA, USA).