2.1. Antibodies and reagents
Total Exosome Isolation Reagent (Invitrogen, 4478359), RPMI 1640 (Himedia, AT120), Dulbecco’s Modified Eagle Medium (DMEM) (Himedia, AT007), Fetal bovine serum (FBS) (10270), Exosome depleted Fetal Bovine Serum (Gibco), Hydrogen peroxide 30% (Merck,107209), Phosphate Buffer Saline (Himedia, TS1006), Dynabeads Protein A (Invitrogen, LT-02241), [3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) purchased from SRL (India), Extraction buffer (Isopropanol Merck DA9P690159, TritonX-100 807428, Hydrochloric acid), Paraformaldehyde (Himedia, RM3660), Tween-20 (Amresco, CAS 9005-64-5), DAPI (Vectashield, H-1200-10), Senescence Beta-Galactosidase Staining Kit (Cell Signalling Technology, CAS 9860S), Low Melting Point Agarose (SRL, CAS 9012-36-6), Electrophoresis buffer (1M sodium EDTA, 300mM NaOH), Neutralizing buffer (0.4 M Tris, pH-7.5), EtBr (20µg/mL), Lysis buffer (1% Triton X-100, 50 mM NaCl, 50 mM NaF, 20 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 0.2 mM Phenylmethanesulfonylfluoride fluoride (PMSF), 0.5% NP-40, protease and phosphatase inhibitor), Glycine (CAS 56-40-6), Tris base (CAS 77-86-1), SDS (CAS 151-21-3), Acrylamide/bis-acrylamide (79-06-1), polyvinylidene difluoride membrane, 5% non-fat dry milk, 0.05% Tween-20, 20mM Tris-Cl, pH 7.6 (TBS-T), Protease inhibitor cocktail (M250-1 ml), Phosphatase inhibitor cocktail II (GX-0211AR) were from Genetix, India, Bovine serum albumin (BSA) (Sigma Aldrich A9418), Bradford’s reagent (Sigma Aldrich B6916), Flotilin 1 rabbit mAb (Cell Signalling Technology, D2V7J), Annexin V (Cell Signalling Technology, 8555T), CD9 Rabbit mAb (Cell Signalling Technology, D801A), CD63 rabbit polyclonal antibody (Santa Cruz Biotechnology, sc-15363), PTEN rabbit monoclonal antibody (Abcam, ab32199), GAPDH rabbit polyclonal antibody (Bio-Bharati LifeScience, BB-AB0060), Akt rabbit polyclonal antibody (Cell Signalling Technologies, 9272s), Phospho-Akt rabbit monoclonal antibody (Cell Signalling Technologies, 4058s), H3K9me3 rabbit polyclonal antibody (Abcam ab8898), γ-H2AX Rabbit polyclonal antibody (Santa Cruz Biotechnology, sc-101696). Anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (HRP, 1:1000, Bangalore Genei, India) and anti-rabbit IgG conjugated with FITC and anti-mouse IgG conjugated with Texas Red (1:200, Santa Cruz Biotechnology) were used as secondary antibody.
2.2. Cell culture and treatment
PC-3 (Human prostate carcinoma cells), A549 (Human lung carcinoma cells), HeLa (Cervical carcinoma cells) and WI-38 (Normal lung fibroblast cells) were purchased from the National Center for Cell Science, (Pune, India) and maintained at 37°C, 5% CO2 and 95% relative humidity (RH) in RPMI 1640 and DMEM media respectively, supplemented with 10% FBS and/or 10% Exosome depleted FBS, penicillin-streptomycin (100 units/ml) and, amphotericin-B (anti-fungal). Cells were seeded overnight in two 60 mm culture plates in media supplemented with 10% FBS. Then, media of both the plates were withdrawn and fresh media supplemented with 10% exosome depleted FBS were added to the plates. One of the plates was treated with hydrogen peroxide (H2O2) (150 µM for 48 h) and the other was kept as untreated.
2.3. Isolation of Exosome
Exosome isolation was done using kit reagent. The cell culture medium was collected according to the manufacturer’s protocol. After the isolation, the exosome pellets were finally dissolved in 1X PBS and stored at -80˚C for further uses.
2.4. Dynamic Light Scattering (DLS)
The particle diameter and size distribution of the isolated samples were measured by DLS instrument (ZetaSizer-HT Malvern). The Z-average values and the particle dispersion index (PDI) values were obtained for comparative study of the sizes of the isolated samples.
2.5. Scanning Electron Microscopy (SEM)
Pellets containing exosomes from healthy cells and senescent cells were fixed with 2% paraformaldehyde for 10 mins. The samples were then added to cleaned silicon chips, followed by sonication in ethanol for 5 mins and immobilized after drying under a laminar airflow. To make the surface conductive, a coating of 2–5 nm gold-palladium alloy was applied by sputtering (Argon as gas for plasma) before imaging by scanning electron microscopy (Inspect FEI, Netherlands). Analysis of exosome sizes was done using the SEM images via ImageJ software.
2.6. MTT assay
The inhibition of cell growth was measured by MTT assay. In brief, the human cell lines PC3, WI38 respectively seeded in 12 well plates. PC3 cells was treated with exosomes (100µg/mL) isolated from mock treated A549 and Hela cells respectively and treated with exosomes (100µg/mL) isolated from A549 cells treated with H2O2.Similarly WI38 cell was treated with exosomes (100µg/mL) isolated from mock treated and H2O2 treated A549cells. In another set PC3 cells was treated with exosomes (100µg/mL) isolated from mock treated and H2O2 treated PC3 cells. MTT reagents are incubated for 3 h at 37 ̊C. The resulting formazan crystals were then dissolved in MTT solubilization buffer and the absorbance were taken with a UV–vis spectrophotometer (Hitachi) at 570.
2.7. Senescence associated (SA)-β-galactosidase staining assay
A549 cells were seeded overnight on cover slip. Next day, cells were washed with 1X PBS and were replaced with fresh medium. One set of cells were kept as control and the other set was treated with H2O2 (150µM) and incubated for 2 h. After the completion of the incubation period the cells were stained with a senescence associated beta-galactosidase staining kit (Cell Signaling Technology, CAS 9860S). Finally, the stained coverslips were mounted on grease free slides using 50% glycerol. Then, the slides were examined under a bright field of fluorescent microscope for detecting the senescent cells under 10 X magnification.
2.8. Fluorescence microscopy
A549 cells were seeded overnight on cover slip. The next day, cells were washed with 1X PBS and media were replaced by fresh medium. Then, one set of cells were kept as control and the other set was treated with H2O2 (150µg/mL) and incubated for 48 h. The cells, after being incubated were fixed with 4% paraformaldehyde solution for 15 min and permeabilized with 0.2% Triton X-100 at 4˚C, for another 15 min. The cells were again washed followed by addition of 5% blocking solution (0.5% FBS in 1x PBS) for 1 h at room temperature followed by overnight incubation with H3k9me3 and γH2AX antibody in wash buffer (0.5% FBS and 0.05% Tween 20 in 1x PBS) at 4°C. Next day, the cells were washed with wash buffer and followed by addition of anti-rabbit IgG conjugated with FITC and anti-mouse IgG conjugated with Texas Red antibodies for 1 h at room temperature (dark condition). After washing, cells containing coverslips were mounted with the mounting solution containing DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, USA) and examined under a fluorescence microscope (100X objectives, Leica, Germany).
2.9. Western blotting
Whole cell lysates were prepared by using cell lysis buffer (50 mm Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP-40, 1 mM dithiothreitol, 2 µg/ml aprotinin, 2 µg/ml leupeptin, protease and phosphatase inhibitor). The protein concentrations of the isolated exosomes and whole cell lysates were also evaluated by Bradford assay. The cellular extracts and exosomes were solubilized in a protein loading buffer, boiled for 5 min, and electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were then transferred to methanol activated polyvinylidene difluoride (PVDF) membrane. The membrane then is blocked in 5% non-fat dry milk in 0.05% Tween-20 in 20 mM Tris–Cl, pH 7.6 (TBS-T) for 1 h at room temperature. After overnight incubation with appropriate primary antibody, the membrane was washed with TBST followed by re-incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Proteins were detected by Advansta ECL western blotting detection reagent. The intensity of each band was measured by ImageJ software.
2.10. Comet Assay
PC3 cells were seeded in 35 mm cell culture plates overnight. Next day, keeping one set without being treated and other two sets were treated with exosomes derived from mock treated and H2O2 treated A549 cells, respectively for 48 h. Then, the cells were trypsinized and centrifuged. The pellets were washed in 1X PBS. PC3 cell pellet was mixed with 37°C 0.8% low-melting point agarose and transferred to normal-melting point agarose coated slides. The slides were mounted with cover slip and incubated at 4°C for solidification. Then, the slides were immersed in lysis buffer (2.5 mM NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Triton X-100) for 1 h at 4˚C. After washing with neutralizing buffer (0.4 M Tris, pH-7.5), the slides were immersed into electrophoresis buffer within an electrophoresis tank. Electrophoresis was carried out at 25V and 300 mA for20 min. After which, the slides were washed with a neutralizing buffer for 40 min. After washing the slides were stained with EtBr solution and kept in the dark for 20 min. The stained slides were washed with 1X PBS and were finally mounted with cover slips by using 50% glycerol as mounting media and scored using a fluorescence microscope (Leica, Germany). % of tail DNA (DNA fragment percent in the tail) were scored with the help of Komet Assay Software 5.5 at 40X magnification.
2.11. Cell damage detection
a) Analysis of nuclear morphology by DAPI staining
Cells were seeded on cover slips. Next day, keeping one set as control, the other set cells were treated and then incubated for 48 h. Then, the cells were washed with 1X PBS for 2–3 times and the cell containing coverslips were mounted on grease free slides with DAPI stain and finally observed under 100 X objective of fluorescent microscope.
b) Cell staining with Hoechst dye
Cells were seeded on cover slips. Next day, cells were treated and incubated for 48 h keeping one set as control. The cells were washed with 1X PBS for 2 to 3 times. Then the cells were stained with 1X Hoechst dye solution, followed by 15 mins of incubation in dark condition, at room temperature. After washing with 1X PBS for 1 to 2 times, cells were mounted with 50% glycerol on grease free slides and were examined under fluorescent microscope at 100 X magnification.
2.12. Cell cycle analysis
PC3 cells were seeded in 60 mm cell culture plates overnight. At 24 h after seeding, keeping one set without being treated and other two sets were treated with 100 µg/mL of exosomes derived from mock treated and H2O2 treated A549 cells, respectively for 48 h. Then, the cells were trypsinized and centrifuged resulting in the formation of pellets. The pellets were washed with 1X PBS and fixed with chilled 70% ethanol and kept for overnight at -20°C. Prior to stain with 50 µg/mL propidium iodide (PI, Invitrogen), the cells were incubated for 30 mins with 100 µg/mL of RNAse A (SRL, India) at 37°C. The cell cycle was analysed with flow cytometry.
2.13. Statistical analysis
A student’s t-test was used to calculate the statistical significance of changes between the groups. P ˂ 0.05 and 0.005 was considered as statistically significant. Data analysis was performed using the Origin Pro v.8 software (Origin Lab).