The proteins were detected by incubation with appropriate primary antibody followed by horseradish peroxidase-conjugated secondary antibody (Pierce) and the ECL reagent (Pierce) and exposed to a film, or images were captured using a ChemiDoc Touch Imaging System (Bio-Rad) and processed using ImageLab software. overnight at 4C, and then centrifuged for 1?hr. The pelleted exosomes were then resuspended in 200?L of PBS or were lysed in RIPA buffer and then quantified by a bicinchoninic acid (BCA) assay using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, China) according to manufacturers instructions. Exosome protein content was determined by calibration against standard curve, which was prepared by plotting the absorbance at 562?nm versus BSA standard concentration. Exosome Size Analysis Exosome size distribution analysis was carried out using the qNano system (Izon, Christchurch, New Zealand). Izons qNano technology (http://izon.com) was employed to detect extracellular vesicles passing through a nanopore by way of a single-molecule electrophoresis.46 In practice, it enables accurate particle-by-particle characterization of vesicles from 75 to 150?nm in size of exosomes, without averaging the particle sizes. Purified exosomes were diluted to 1 1:10 in PBS with 0.05% Tween 20, vigorously shaken, and measured by using an NP150 (A45540) nanopore aperture according to the manufacturers instructions. Data processing and analysis were carried out around the Izon Control Suite software v3.3 (Izon Science). qRT-PCR for miRNA Total RNAs from cells and liquid exosomes were isolated using TRIzol reagent (Thermo Fisher Scientific) PIK3CD and TRIzol LS reagent (Thermo Fisher Scientific) according to the respective manufacturers instructions. The procedure was performed as explained.22 The miRNA tested were reverse-transcribed from 50?ng total RNA in duplicate by specific stem-loop primer as explained in the TaqMan miRNA BAY 41-2272 reverse transcription kit (Applied Biosystems). The expression of miRNA was determined by real-time PCR using TaqMan Universal Master Mix II kit purchased from Applied Biosystems. miRNA copy number was normalized by comparison with cellular 18?s rRNA. The primers of miR401 were designed according to Chen et?al.47 and synthesized by Ige Biotechnology. The sequences are as follows: miR401 stem loop primer, 5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCGT-3; forward primer, 5-TGCTGGCGAAGAGGATGC-3; reverse primer, 5-CCAGTGCAGGGTCCGAGGTA-3; probe, 5-(6-FAM)CTGGATACGACCCTCGTC(MGB)-3. BAY 41-2272 Immunoblot Assays Purified exosomes were harvested and lysed with a RIPA lysis buffer (Beyotime) supplemented with 1?mM protease inhibitor PMSF (Beyotime) and phosphatase inhibitor (Beyotime). Cell lysates were warmth denatured, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore). The proteins were detected by incubation with appropriate primary antibody followed by horseradish peroxidase-conjugated secondary antibody (Pierce) and the ECL reagent (Pierce) and exposed to a film, or images were captured using a BAY 41-2272 ChemiDoc Touch Imaging System (Bio-Rad) and processed using ImageLab software. The densities of corresponding bands were quantified using ImageJ software. Computer virus Titration Cells were seeded in 6-well plates or 24-well plates at densities of 1 1??106 cells per well or 2.5? 105 cells per well, respectively, for 24?hr and then exposed to BAY 41-2272 0.01 or 0.1 PFU of HSV-1(F) per cell. The cells were harvested at 3, 6, 12, 24, and 48?hr post-infection or at indicated time point. Viral progeny was titrated on Vero cells after three freeze-thaw cycles and brief sonication.48 Author Contributions L.W., X.C., B.R., and G.G.Z. designed research; L.W., X.C., and X.Z. performed research; X.C., B.R., and G.Z. analyzed data and published the paper. Conflicts of Interest The authors declare no discord of interest. Acknowledgments These studies were supported by grants from the National Nature Science Foundation of China (NSFC 81472826 and NSFC 31600137), Guangzhou Science Technology, Innovation Commission rate Project (201504010016 to Guangzhou Medical University or college), and Developmental Funding of Dapeng New District (KY20160302 to Shenzhen International Institute for Biomedical Research)..