Fv3000 confocal microscope

PKH26 kit (Sigma, USA) was used to label exosomes. The PKH26 working solution was added to the exosomes resuspended in Diluent C according to the instructions. After incubation, ultracentrifugation (100 000 rpm) was used to remove the free PKH26 working solution. Subsequently, exosomes resuspended in PBS were added to the cells for co-culture. After the incubation period, all medium was discarded. 4% paraformaldehyde was used for fixation. DAPI staining solution was used to stain nuclei. The OLYMPUS FV3000 Confocal Microscope was used for fluorescence detection.

MDA-MB-231 cells were seeded on glass coverslips and cultured at 37 °C overnight. Alexa Fluor 647 labeled RNA nanoparticles were incubated with cells at a final concentration of 100 nM for 4 h at 37 °C. After washing twice with PBS buffer, cells were fixed with 4% formaldehyde and washed again, followed by treatment with 0.1% Triton X-100 (Sigma-Aldrich) in PBS buffer for 5 min and subsequent cytoskeleton staining with Alexa Fluor 488 phalloidin (ThermoFisher Scientific) for 30 min at room temperature. After rinsing with PBS buffer, the cells were mounted with ProLong^@ Gold Antifade Reagent (Life Technologies Corp.) containing DAPI for cell nucleus staining and assayed on Olympus FV3000 confocal microscope (Olympus Corp.). Data were collected using Fluoview FV31S-SW.

Cells grown on coverslips in a 6-well plate were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by permeabilization with chilled methanol on ice for 10 min. Cells were then blocked with 1% BSA and 10% goat serum in PBS for 1 h, followed by incubation with primary antibodies at 4℃ for overnight. Next, fluorescence-tagged secondary antibodies were added and incubated at room temperature for 1 h together with DAPI for nuclear staining. Samples were mounted on glass slides using fluorescence mounting medium (DAKO, Carpinteria, CA, USA) and examined under an Olympus FV3000 confocal microscope (Olympus Co., Tokyo, Japan) with 60x/1.40 oil objective lens. For images analysis and cell counting, the ImageJ software (https://imagej.nih.gov/ij/download.html) was used.

The corneal cryosections were permeabilized with 0.1% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 15 min and blocked in PBS supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% bovine serum albumin (BSA; Thermo Scientific, Waltham, MA, USA) at 37 °C for 1 h. Hybridization with primary antibodies against Cytokeratin 15 diluted to 1:500 (MA5-15567; Thermo Fisher Scientific, Waltham, MA, USA) was performed overnight at +4 °C. After several washes, the Anti-Mouse IgG H&L antibodies conjugated with goat anti-mouse Alexa Fluor 555 diluted to 1:500 (ab150114; Abcam, Cambridge, UK) were added for 60 min at room temperature. The nuclei were counterstained with DAPI (1 µg/mL; Thermo Fisher Scientific, Waltham, MA, USA). Sections were observed using single scans made using an OLYMPUS FV3000 confocal microscope (Olympus, Center Valley, PA, USA). ImageJ (v.2.1 software) was used to process and analyze the obtained images.

Immunofluorescence assay was performed to determine the effects of different treatment on cell commitment towards differentiation. Briefly, myoblast cells grown on glass coverslips were firstly washed with PBS for three times and then fixed with 4% formaldehyde for 20 min and permeabilized in 0.2% Triton X‐100 for 15 min. Thereafter, the cells were blocked non‐specific binding by incubating with 5% bovine serum albumen (BSA) in PBS for 1 h and incubated with primary antibodies diluted in 1% BSA at 4°C overnight, followed by incubation with the corresponding fluorophore‐conjugated secondary antibodies (1:400, cat. no. BA1101/BA1032, FITC/Cy3‐labelled IgG, Boster, China) in dark for 1 h at room temperature. After washing with PBS for five times, cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 20 min in dark. The primary antibodies used were as follows: PAX7 (1:200, cat. no. sc‐81648, Santa Cruz), MyoD (1:300, cat. no. NB100‐56511, Novus), MyoG (1:500, cat. no. M5815, Sigma‐Aldrich) and MyHC (cat. no. MF20, 1:500, DSHB). The coverslips were imaged using Olympus FV3000 confocal microscope (Olympus, Tokyo, Japan). All immunostaining was performed in three independent experiments, and at least six views were captured from each cell well.