Ab108950

For extraction of BMSC-derived exosomes, after 48 h of transfection, the supernatant was collected, centrifuged and resuspended in 200 μl PBS as described elsewhere [18 (link)]. After fixed with 2% paraformaldehyde and loaded on parafilm, the exosome samples were observed under a transmission electron microscopy (TEM; JEM-1400PLUS, Japan) at 100 KV and the nanoparticle-tracking analysis (NTA) software was used to evaluate the size of exosomes.
A BCA protein assay kit (Beyotime, China) was used to measure the protein concentration of the exosomes. The identification of exosomes was performed by Western blot assay for analysis of exosome markers CD9, CD63, CD81 and TSG101. Briefly, exosome proteins were separated by 10% SDS-PAGE, transferred onto PVDF membrane and then incubated with primary antibodies of anti-CD9 (ab92726, Abcam) and anti-CD63 (ab108950, Abcam), anti-CD81 (ab109201, Abcam) and anti-TSG101 (ab125011, Abcam) at 4 °C overnight, following with incubation of corresponding secondary antibodies at 37 °C for 45 min. Proteins were visualized on an Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE). For treatment of tenocytes, the extracted exosomes (20 μg/ml) were added into the plates and the control cells were untreated.

Total protein was collected using Radio-Immunoprecipitation assay cell lysis buffer, and the protein concentration was determined using the BCA kit. The protein was then separated by 6% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk at 20°C for 1 h and incubated with the primary antibodies prepared in 5% bovine serum albumin at 4°C overnight, followed by incubation with the secondary antibodies in 5% non-fat milk at 20°C for 1.5 h. After that, the membranes were soaked in enhanced chemiluminescence reagent (WBKLS0100, Millipore, Corp. Billerica, MA, USA) and then exposed for film development. The protein bands with immunoblots were analyzed, and the relative protein level was calculated based on the gray value ratio of target bands and internal references. Three independent experiments were performed. The antibodies used were TSG101 (1:2000, ab125011, Abcam, Cambridge, UK), CD9 (1:2000, ab92726, Abcam), CD63 (1:100, ab108950, Abcam), Bax (1:5000, ab32503, Abcam), Bcl-2 (1:2000, ab196495, Abcam), β-actin (1:11,000, ab6276, Abcam) and HDAC1 (1:2000, ab7028, Abcam) and HRP-labeled secondary antibodies goat anti-rabbit IgG (1:50,000, ab205718, Abcam) and goat anti-mouse IgG (1:10,000, ab205719, Abcam).