Indirectly loaded exosomes alone can induce potent OVA-specific primary antibody responses. BMDCs were stimulated with LPS on day 6, and exosomes were harvested on day 8. BALB/c mice were primed intravenously with 50 μg OVA per mouse alone or together with 10 μg LPS, Alum (1:1 ratio with OVA), 50 μg OVA-Exo, or Pep-Exo or only with 50 μg OVA-Exo. Sera were collected 7 days after priming, and the presence of OVA-specific (A) IgM and (B) IgG antibodies was detected by ELISA using serial dilution of the sera. Results are expressed as optical density at 1:100 serum dilutions. Individual data and mean are presented (n = 7 per group, for Alum group n = 5). (C) A total of 30 μg OVA-Exo and Exo or 1 μg native OVA protein was separated on 8% to 16% SDS-acrylamide gel, transferred to polyvinylidene difluoride membrane, and incubated with OVA-specific immune sera (1:500 dilutions), followed by incubation with horseradish peroxidase–conjugated secondary antibodies, and detected by chemiluminescence kit. (D) A total of 30 μg OVA-Exo was coated on anti-CD9–coated latex beads, treated with OVA immune sera (open histogram) or preimmune sera (solid histogram), followed by addition of PE-conjugated anti–mouse Ig, and then analyzed by flow cytometry.