Data are representative of three separate analyses. soluble CX3CL1. Therefore, our study suggests that CX3CR1 is a novel and ligand-competent exosome receptor. culturing and activating primary T cells as previously described [6]. In brief, after eliminating erythrocytes by using ammonium-chloride-potassium (ACK) buffer (Thermo Fisher Scientific, Waltham, MA, USA), the lymphocytes were subjected to resuspension in RPMI1640 (Nacalai, Kyoto, Japan) containing 10% exosome-depleted fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX, USA) and penicillin/streptomycin (Nacalai). The cells were then plated on culture dishes coated with anti-CD3 (3?g/ml) and anti-CD28 (3?g/ml) antibodies (BD Biosciences, San Jose, CA, USA) in a 37?C incubator with 5% CO2 supply. T MHY1485 cells activated for 48?h were moved onto antibody-uncoated dishes and further incubated for 72?h in the same culture media supplemented with interleukin-2 (1?ng/ml) (R&D Systems, Minneapolis, MN, USA). As mentioned above, to eliminate extracellular vesicles (EVs) including exosomes in FBS, FBS was centrifuged at 76,000?for 18?h at 4?C using polypropylene centrifuge tubes (Beckman Coulter, Brea, CA, USA), a swing bucket rotor (SW 28 Ti, Beckman Coulter) and an L60 Ultracentrifuge (Beckman Coulter) and the supernatant solution was filtered through 0.22-m filter units (Merck, Darmstadt, Germany). This filtrated FBS was considered to be exosome-depleted and was used in this study for exosome isolation from the cells. Several mouse tumor cell lines including TK1, CT26.WT, EL4, LTPA, and B16F10?cells were obtained from ATCC (Manassas, VA, USA). RAW264.7 (mouse macrophages) and THP-1 (human monocytes) were also from ATCC. MLO-Y4 (osteocyte) and MLO-A5 (osteoblast) cells were purchased from Kerafast (Boston, MA, USA). All cells were cultured, according to the manufacturers instructions, for 48?h in RPMI1640, DMEM (Nacalai), or MEM (Thermo Fisher Scientific) media supplemented with 10% EV-depleted FBS and penicillin/streptomycin. 2.2. Mice C57BL/6J mice (8C11 weeks old) were purchased from CLEA Japan (Tokyo, Japan) and maintained at the Experimental Animal Facility of Mie University. Experimental animal protocols were approved by the Ethics Review Committee for Animal Experimentation of Mie University (Approval number: #27-6-2). Spleens, bone marrows, and lungs were separated from the mice and used to isolate cells by using a mechanical dissociation with Falcon 40-m cell strainers (Corning, Glendale, AZ, USA). The media collected in the cell isolation procedure were used to isolate exosomes (see section 2.3. for detail). 2.3. Isolation and characterization of exosomes Exosomes were isolated as previously described with minor changes [6,27,28]. Briefly, culture media were centrifuged at 1000?for 10?min MHY1485 at 4?C to remove cells. The supernatant was spun at 2000?for 20?min at 4?C to eliminate apoptotic bodies. The supernatant was centrifuged in an L60 Ultracentrifuge (Beckman Coulter) at 24,000?for 20?min at 4?C. The supernatant was then subjected to a second centrifugation at 110,000?for 2?h at 4?C. The pelleted exosomes were subsequently suspended in phosphate-buffered saline (PBS) buffer (Nacalai). In turn, this exosome solution was passed through a 0.22-m filter unit and spun at 110,000?for 2?h at 4?C. The pellet (exosomes) was suspended in PBS buffer. The concentration was measured with a bicinchoninic acid protein assay kit Retn (Thermo Fisher Scientific). The particle size was characterized by using a dynamic light scattering (DLS) device (Horiba, Kyoto, Japan). 2.4. Exosome conjugation with microbeads The exosomes were conjugated to 4-m latex beads for efficient detection and then stained with fluorescently labeled monoclonal antibodies as previously shown [6]. In brief, after standardizing all different exosomes equally at 0.5?mg/ml in PBS, the exosomes (5?g) were conjugated to microbeads (10?l) (Thermo Fisher Scientific) in 1?ml of PBS by incubating the mixture for 2?h using a tube rotator and then blocked by an incubation with 100?mM glycine for 30?min. The exosomes were washed three times with PBS comprising 0.5% bovine serum albumine (Sigma, St. Louis, MO, USA). The same amounts of exosome samples (5?g) coupled to 10?l latex beads were subjected to circulation cytometry below so that the expressions are detected feasibly at similar amounts of exosomes. In some experiments, 1?ml of PBS, 1?ml of EV-depleted FBS, or 1?ml of FBS were conjugated with 10?l latex beads as done with the same methods as exosomes and then assessed for any expressions via using circulation cytometry. 2.5. Circulation cytometry analysis Antibodies to CD9 (HI9a), CD63 (NVG-2), CD63 (H5C6), CD81 (Eat-2), CCR9 (9B1), CXCR4 (L276F12), and CX3CR1 (SA011F11) were purchased from BioLegend (San Diego, CA). Isotype settings including Rat IgG2a, Rat IgG2b, Armenian Hamster IgG, Mouse IgG2a, and Mouse IgG1 were also from BioLegend. The antibody to CD9 (KMC8) was from BD Biosciences. The antibodies to CCR7 (4B12), CCR10 (248918) were acquired from R&D Systems. The cells or microbead-conjugated exosomes were stained with the fluorescently labeled antibodies, washed twice with PBS comprising 2% FBS and 2?mM ethylenediaminetetraacetic acid (EDTA) (Wako, Osaka, Japan), and MHY1485 analyzed by using BD Accuri C6 circulation cytometer and software (BD Biosciences). For this method of microbead conjugation of exosomes,.