The alkaline phosphatase (ALP) activity of EMVs was assayed using ALP colorimetric

kit (AnaSpec, Fremont, CA). Briefly, 50-μg vesicles were incubated with a colorimetric substrate, para-nitrophenyl phosphate, and the conversion of para-nitrophenyl phosphate to p-nitrophenol on release of phosphate ions was monitored at 405 nm. The protein concentration of the EMV samples was measured by Bradford assay (Bio-Rad Laboratories, Hercules, CA). For detection of mCherry fluorescence on EMVs derived from mCherry-labeled, 143B luciferase–expressing, puromycin-resistant cells, EMV suspensions were examined microscopically using the Olympus (Center Valley, PA) IX71 inverted fluorescent microscope equipped with a xenon arc lamp and monochromatic complementary metal oxide semiconductor camera. In addition, flow cytometric this website Obeticholic Acid price data were acquired on EMV suspensions using the BD LSR II flow cytometer integrated with FACSDiva software (BD Biosciences,

San Jose, CA, USA). For TEM, 143B EMV pellets were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetraoxide (OsO4), dehydrated, embedded in epon resin, and cut into ultrathin sections. The sections were stained with uranyl acetate and lead acetate before mounting on EM grids. The sections were examined and photographed using a JEM 1400 electron microscope (JEOL USA, Inc., Peabody, MA, USA) (80 kV). To determine the biochemical composition of the 143B EMV cargo, Western blot analyses were performed according to the previously described method [30]. 143B EMVs

were homogenized in Tris lysis buffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM PMSF, and 1 mM DTT). Crude lysates of 143B cells (12.5-25 μg) and EMVs (25-40 μg) were denatured in sodium dodecyl sulfate sample buffer, electrophoresed on 12% denaturing polyacrylamide gels, and visualized by Ponceau stain. For immunoblot analysis, the Clostridium perfringens alpha toxin proteins from the gel were transferred on to a polyvinylidene fluoride (PVDF) membrane and incubated with the following primary antibodies: anti–MMP-1 and anti–MMP-13 (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); 200 μg/ml each) at 1:200, anti-CD-9 (SBI: System Biosciences (Mountain View, CA, USA); 0.25 mg/ml) at 1:1000, and anti-RANKL and anti–TGF-β (GeneTex (Irvine, CA, USA); 1 mg/ml) at 1:1000 dilution. Detection of the immunostained bands was done by ECL chemiluminescence detection system (Thermo Scientific, Rockford, IL). Image acquisition was done using LabWorks Image Acquisition and Analysis Software 4.6.00.0 (UVP Bioimaging Systems, Upland, CA) and Image Lab software for the ChemiDoc MP system (Bio-Rad Laboratories) at incremental exposure time frame of 15, 30, 60, 180, and 300 seconds.