Anti cd63 antibody

RPMI-1640 and DMEM medium and foetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY). Trizol, PrimeScript RT Master Mix and SYBR green PCR master mix were purchased from Takara (Shiga, Japan). Antibodies against LC-3I/II, p62, VPS34, and HDAC6 were purchased from Cell Signaling Technology (Danvers, USA), antibodies against GFP, GRP78 and acetyl-lysine were from Abcam (Cambridge, UK), and anitbodies against β-tubulin, β-actin and GAPDH were from Abmart (Shanghai, China). The anti-CD63 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, USA), and the anti-FITC-, -TRITC-, -Cy5- and -HRP-conjugated secondary antibodies as well as ER-Tracker and Lyso-Tracker were obtained from Invitrogen (Carlsbad, USA). Sodium butyrate, vorinostat (SAHA), tubastatin A and 3-methyladenine were obtained from Cayman Chemical (MI, USA). Anti-FLAG M2 mAb and anti-FLAG M2 agarose were obtained from Sigma (St. Louis, USA).

Isolated exosomes were analyzed according to their surface tetraspanin proteins using a modified protocol (Barranco et al., 2019 (link)). Conjugated antibodies against CD9, CD63, and CD81 were used to label the exosomes. 50 μL of Exosome MicroBeads was added to the exosome sample, vortexed and incubated for 1 h at room temperature. Following incubation exosome bound beads were further washed in PBS/1% BSA, blocked with 10% BSA, and stained with Anti-CD9 [CD9 (Santa Cruz Biotechnology, (C-4):sc-13118) conjugated with AlexaFlour@488, Anti-CD63 Antibody (Santa Cruz Biotechnology, MX-49.129.5: sc-5275) conjugated with AlexaFlour@647, and Anti-CD81 Antibody (Santa Cruz Biotechnology, (1.3.3.22): sc-7637] conjugated with AlexaFlour@546, all in concentration 1 µg/mL. The stained sample was incubated for 1 h at room temperature then washed and resuspended in FACS buffer for further analysis using BD FACS Calibur.
The size and the size distribution of both Ex and LUT-Ex were determined using Zetasizer (Malvern Instruments, UK) after their appropriate dilution with water. Additionally, their morphology was examined using transmission electron microscopy (TEM) (Jeol, JEM-100 CX electron microscope, Tokyo, Japan) after being stained with 1% w/v phosphotungstic acid. Their total protein content was quantified using the BCA protein assay kit (Sigma-Aldrich, USA).

Total protein of urinary exosomes was extracted with Urine Exosome RNA Isolation Kit, Appendix A (Biotek Corporation, Thorold, ON, Canada) according to manufacturer's instructions. Equal amounts of protein lysates were subjected to Western blotting analysis by using anti-CD63 antibody (SantaCruz Biotechnology, Dallas, TX, USA), according to standard protocols.