Cell cycle analysis: Flow cytometry and Western blotting of the phosphorylated and non-phosphorylated protein of Retinoblastoma (pRb)

Vero cells (1x10⁵) were synchronized according to the method described previously by Osuna et al. (1984) [27]. Cells were seeded in 6-well plates with a culture medium with 25 mM thymidine for 12 h, when the medium was replaced with MEM + 10% IFCS. Afterwards, cells were washed with MEM and 1 h later, EVs were added directly to each well. One hour after this EV-cell contact, cells were washed and maintained for 2 and 8 h. Afterwards, the culture medium of the corresponding wells was removed, the cells were washed with PBS, fixed with 70% cold ethanol and incubated with a solution (0.2 M Na₂HPO₄, 0.1 M citric acid, pH 7.8) for 15 min at 37°C. Cells were then centrifuged, washed with PBS and resuspended in 250 mL of a solution of propidium iodide (40 mg/mL) and RNAse (100 mg/mL) for 30 min at 37°C in the dark, according to Carrasco et al. (2014) [28]. Finally, the samples were analysed in a FACS Calibur (BD Biosciences, San Jose, CA, USA) flow cytometer. The results were analysed with FlowJo software (v 7.6.5, Tree Star, Inc.).

Phosphorylation of the protein Rb after the incubation of cells with EVs was evaluated by immunoblotting. Briefly, 1x10⁵ cells were grown in 6-well plates for 24 h. Cells were washed with MEM and incubated with EVs for 5, 10, 30 and 60 min. After this session, the cells were washed with PBS and lysed in RIPA lysis buffer with a protease inhibitor cocktail (Roche, Switzerland) for 15 min. Cells were harvested with a cell scraper and centrifuged at 14,000 xg for 10 min at 4°C. Supernatants were transferred to new Eppendorf tubes and stored at -20°C. The protein from cell lysates was quantified using the Bradford reagent (Sigma, USA) and 90 μg of protein from cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and blocked overnight with 5% non-fat milk in PBS-0.1% Tween 20. Rb (1:2,000) and phospho-Rb (1:1,000) (Cytoskeleton, USA) primary antibodies (Sigma, USA) were incubated overnight at 4°C. Tubulin antibody (1:5,000) (Cytoskeleton, USA) was used as the loading control. The membranes were washed with PBS-0.1% Tween 20 and incubated for 1 h with goat anti-mouse IgGs conjugated with peroxidase (1:1,000) (Dako Agilent Pathology Solutions, USA) in the case of Rb, goat anti-rabbit IgGs conjugated with peroxidase (1:2,000) in the case of phospho-Rb (Dako Agilent Pathology Solutions, USA), and rabbit anti-sheep IgGs conjugated with peroxidase (1:5,000) (Dako Agilent Pathology Solutions, USA) in the case of tubulin. The reaction was also visualized using Clarity ECL Western substrate (BioRad, Spain) in a ChemiDoc Imaging system (BioRad, Spain).