Anti human cd9

Isolated EVs were resuspended in RIPA buffer (1% Nonidet p-40, 0.1% SDS, 0.1% sodium deoxycholate, protease inhibitor cocktail (Sigma-Aldrich), in PBS pH 7.5) and 2 μg of proteins for each sample were loaded on 4–12% NuPAGE Bis-Tris gel (Life Technologies, Carlsbad, CA, USA). Electrophoresis was performed and proteins were blotted on a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated overnight at 4 °C with specific primary antibodies for: anti-human CD9 (1:1000 dilution, Abcam, Cambridge, UK), anti-human CD63 (1:1000 dilution, Thermo Fisher Scientific, Waltham, MA, USA), anti-human CD81 (1:5000 dilution, BD Biosciences, San Jose, CA, USA), anti-human syntenin-1 (1:1000 dilution, Abcam), anti-flotillin-1 (1:10,000 dilution, Abcam). A specific HRP-conjugated secondary antibody (1:2000 dilution, Cell Signaling Technology, Danvers, MA, USA) was used for the detection. Positivity was highlighted by providing the substrates for the chemiluminescence reaction of HRP (GE Healthcare, Chicago, IL, USA). Gel running was performed under non-denaturizing conditions for the detection of CD9, CD81 and CD63 and under denatured conditions for other markers.

The hGCs were digested separately by trypsin–EDTA for 3 min and were blown into single cells gently, which were fixed and permeated by the Cytofix/Cytoper Fixation/Permeabilization Solution Kit (BD, USA) following the instructions of the manufacturer. The hGCs were then stained with PE or FITC-conjugated antibodies for anti-human-ki67 (Abcam, USA), anti-human-AMH (Thermo, USA), anti-human-FSHR (Thermo, USA), anti-human-FOXL2 (Thermo, USA), anti-human-CYP19A1 (Abgent, USA), anti-human-Annexin V (Abcam, USA), anti-human-CD9 (Abcam, USA), anti-human-CD63 (Abcam, USA), and anti-human-CD81 (Thermo, USA) or their corresponding isotype control for 30 min at 4 °C as already described. These stained cells were analyzed on a fluorescence-activated cell sorter (Beckman, USA). The experiments were repeated three times, and the results are presented as the fold change ± SD. p < 0.05 determined a significant difference.