Flow cytometry

The ATP concentration of hepatocytes was measured using an ATP assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's protocol. Briefly, the cells were lysed in lysis buffer and centrifuged at 4°C and 12 000 g for 10 minutes. The supernatants were used for ATP detection using a Centro LB960 luminometer (Berthold Technologies, Bad Wildbad, Germany). The concentration of ATP was calculated in accordance with a standard curve and converted into nmol/mg protein.
For the quantitative estimation of mitochondrial membrane potential, hepatocytes were incubated with 10 μmol/L rhodamine 123 (Beyotime Institute of Biotechnology) for 30 minutes in the dark, and the fluorescence intensity of the dye was determined by flow cytometry (Becton‐Dickinson, Mountain View, USA).
The intracellular ROS concentrations were measured using the peroxide‐sensitive fluorescent probe 2′7′‐dichlorofluorescein diacetate (DCFH‐DA) (Beyotime Biotechnology Inc., Nantong, China). The cells were exposed to serum‐free medium containing 10 μmol/L DCFH‐DA and propidium iodide in the dark for 30 minutes and then washed three times with cold PBS. The fluorescence was measured by flow cytometry (Becton‐Dickinson, Mountain View, USA).

The effects of serum starvation and TUDCA-treatment on the cell cycle and apoptosis of nuclear donor cells were examined according to the experimental designs. Cells were digested by 0.25% trypsin in D-Hanks solution for 3 min at 37°C, rinsed 2 times with chilled PBS, fixed in 75% ethanol by freezing for 1 h at -20°C, rinsed once with chilled PBS again, resuspended with 400 μL cold PBS containing 20 μL of RNase A, incubated at 37°C for 30 min, filtered through a 400-mm mesh, mixed with 400 μL of propidium iodide (100 mg/mL) staining solution in the dark, incubated for 1 h at 4°C, and finally examined by flow cytometry (Becton-Dickinson, Oxford, UK). For cell apoptosis analysis, cells were collected by trypsin digestion. After washing 3 times with cold PBS, 400 μL of annexin V (BestBio, BB-4101) and 5 μL of annexin V-EGFP staining medium (BestBio, BB-4101) were added. The cell mixture was mixed slightly, incubated in the dark for 15 min at 4°C, supplemented with 10 μL of propidium iodide (10 mg/mL), incubated for 5 min, and finally examined by flow cytometry (Becton-Dickinson).

Confocal fluorescence microscopy was used to assess the intracellular trafficking of M-EXOs. Cells that had grown on the glass coverslips (pretreated with polylysine) of a six-well plate were incubated with FITC-labeled M-EXOs (FITC-M-EXOs) for 24 h. Following incubation, the cells were washed three times with PBS and fixed in paraformaldehyde for 15 min. Localization of FITC-M-EXOs in cells was visualized using a confocal microscope (Carl Zeiss Microscope Systems, Jena, Germany) with identical settings for each confocal study. To quantify the cellular uptake efficiency, FITC signal uptake rates were detected using flow cytometry (Becton, Dickinson and Company, USA).
To assess the intracellular trafficking of M-EXOs, H22 cells grown on the glass coverslips of a 6-well plate were incubated with FITC-M-EXOs for 4 h. Then, the cells were incubated with culture medium containing 50 nM of LysoTracker blue DND-22 for 0.5 h. The cells were then washed three times with PBS and localization of FITC-M-EXOs in cells was visualized by confocal microscopy with identical settings for each confocal study. In addition, FITC signal uptake rates were detected using flow cytometry (Becton, Dickinson and Company, USA).