Anti cd9

Exosome markers were detected with DELFIA.³⁴ Specifically, ExoDiff or ExoPr0 dilutions or their diluent, phosphate buffer saline (PBS), were loaded in duplicates on high‐binding 96‐well plates (R&D Systems, DY990) and incubated at 4°C. Next day, the plates were washed three times with DELFIA washing buffer before any reagent was added to wells (PerkinElmer, 1244‐114). First, plates were blocked with 1% BSA in PBS for 1 h at room temperature. Then, plates were incubated with anti‐CD9 (1:500), CD63 (1:200) or CD81 (1:500; BD Biosciences, 555 370, 556 019, 555 675). After 2 h, plates were incubated with appropriate secondary antibodies (1:2,000; Abcam, ab98691, ab97073) for 1 h. Afterwards, streptavidin‐europium conjugate in assay buffer (1:1,000) was added to wells for 1 h (PerkinElmer, 1244‐106 and 1244‐30). Finally, plates were washed six times and incubated with enhancement solution, while being shaken at 300rpm for 10 min (PerkinElmer, 1244‐104). The samples were analysed with a PHERAstar plate reader with the following settings: 337 nm excitation, 620 nm detection, 200 μs integration time and 60 μs lag time (BMG Labtech). Fluorescence value of each control, that is PBS incubated with both primary and secondary antibodies was subtracted from sample fluorescence.

EVs derived from ovarian cancer lines were lysed in LIPA lysis buffer (Cell Signaling Technology) containing protease inhibitor (Roche). Western blot analysis was performed as described previously.^[60] The nitrocellulose membrane blots were probed with anti‐CD9 (1:500 dilution, BD Biosciences), anti‐CD63 (1:500 dilution, Ancell), and anti‐CD81 (1:500 dilution, Santa Cruz Biotechnology). The chemiluminescence signal was detected with an Azure 280 imaging system (Azure Biosystems).

Isolated EVs were re-suspended in Laemmli buffer and boiled for 5 minutes at 95 °C. Specifically, 20 µg of EVs were prepared in non-reducing conditions for tetraspanins detection, while 35 µg were used for soluble protein detection. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Nonspecific sites were saturated with 5% (w/v) skimmed milk in Tris-buffered saline: 150 mM NaCl, 20 mM TrisHCl, pH 7.4, and 0.5% Tween 20 (T-TBS). Membranes were incubated overnight at 4 °C with anti-CD9 (1:1000, BD Pharmingen, #555370, San Jose, CA, USA), anti-CD63 (1:2000; BD Pharmingen, #556019, San Jose, CA, USA), anti-Alix (1:500, Santa Cruz, #sc-271975, Santa Cruz, CA, USA), anti-TSG101 (1:500, Novus Bio, #NB200–112, Littleton, CO, USA), anti-DCXR (1:1000, Abcam, #ab110283, Abcam Inc., Cambridge, UK), anti-Calnexin (1:1000, Sigma, #C7617, Sigma-Aldrich, St. Louis, MO, USA) and anti-β-tubulin (1:5000, Santa Cruz, #sc-9104, Santa Cruz, CA, USA). After washing with T-TBS, membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 for 45 min. Immunoreactive bands were revealed by the enhanced chemiluminescence method (ImmobilonHRP substrate, #WBKLS0500, Millipore Corp., Billerica, MA, USA).

EVs were lysed in LIPA lysis buffer (Cell Signaling Technology) for western blot analysis to confirm the characteristic EV biomarkers (CD9, CD63, and CD81). The blots were probed with flowing primary antibodies: anti-CD9 (1:500 dilution, BD Biosciences), anti-CD63 (1:500 dilution, Ancell), and anti-CD81 (1:500 dilution, Santa Cruz Biotechnology). Chemiluminescence was detected using an Azure 280 imaging system (Azure Biosystems). The concentrations and sizes of EVs were determined by nanoparticle tracking analysis using Nanosight NS300 and were found to be 1.22 × 10¹¹ particles/mL (CaOV3), 2.7 ×10¹¹ particles/mL (OV90), 3.0 × 10¹¹ particles/mL (ES2), and 3.8 × 10¹¹ particles/mL (TiOSE4)

The StemPro Differentiation Kit (Life Technologies) was used according to the manufacturer’s recommendations to determine the capacity of hMSCs to undergo adipogenic or chondrogenic differentiation. Characterization of hMSCs and hMSC-microvesicles was performed by fluorescence-activated cell sorting (FACS) utilizing a FACSCalibur™ sorter (BD Biosciences) and analyzed by CellQuest software (BD Biosciences). FITC-conjugated antibodies (anti-CD73, anti-CD90, anti-CD105, anti-CD146, anti-CD14, anti-CD34, anti-CD45, and anti-HLA-DR; BD Biosciences) were incubated with samples for 30 min in PBS containing 0.5% BSA at 4 °C. Mouse isotypic IgG was used as a control.
FACS analysis of hMSC-exosomes was performed using exosome capture beads based on anti-CD63 coupled antibody (Invitrogen Dynabeads Exosome Human CD63 Isolation/Detection Reagent) following the manufacturer’s recommendations. Specific FITC-conjugated exosomal markers (anti-CD9 and anti-CD81; BD Biosciences) were used as above.

D3-EVs and D5-EVs were isolated from a pool of spent media from 50 embryos at different developmental stage and from an equal volume of 10% SSS- or 5% HSA- supplemented fresh media as negative controls and were used for immunoblotting analysis. Ten µg of isolated EVs (by Bradford assay and by NanoDrop8000 measurement) were lysed in reducing sample buffer [0.25 M Tris–HCl (pH 6.8), 40% glycerol, 8% SDS, 5% 2-mercaptoethanol and 0.04% bromophenol blue] and boiled for 5 minutes at 95 °C. For tetraspanins detection, non-reducing sample buffer (without 2-mercaptoethanol) was used. Proteins were resolved by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), electrophoretically transferred to polyvinylidene fluoride membranes, blocked in 5% non-fat powdered milk in TBS-T (0.5% Tween-20) and the membranes were incubated with the following antibodies: anti-ALIX (1:500, Santa Cruz, #sc-271975, Santa Cruz, CA, USA), anti-CD9 (1:1000, BD Pharmingen, #555370, San Jose, CA, USA), and anti-CD63 (1:1000; BD Pharmingen, #556019, San Jose, CA, USA). Protein bands were detected using X-ray film and enhanced chemiluminescence reagent (ECL, Amersham, Buckinghamshire, UK).