Western blotting: Primary antibodies: anti‐CD63 (ab217345, Abcam, USA), anti‐Alix (ab275377, Abcam), anti‐TSG101 (ab125011, Abcam), anti‐Calnexin (ab313243, Abcam). The membranes were developed using ECL kit (Yeasen, China).
TEM: DsEVs sample was examined by TEM (Hitachi TEM system, Japan).
NTA: 10 µL DsEVs suspension was loaded into the sample chamber of ZetaVIEW S/N 252 (Particle Metrix GmbH, Germany). Data analysis was performed with software (ZetaView 8.04.02, Germany).
Detection of the loading rate of TAT47‐57‐MBP87‐99A91 into DsEVs: After co‐culturing the FITC‐labeled peptide with DCs for 24 h, DCs were collected and stained with phalloidin (Actin‐Tracker Red‐594, C2205S, Beyotime) and 4′,6‐diamidino‐2‐phenylindole (DAPI, C1005, Beyotime, China). After the isolation of DsEVs.
DsEVs internalized by DCs and CD4+ T cells in vitro: DsEVs were labelled with the Dil (Solarbio life sciences, China) and then co‐cultured with DCs or CD4+ T cells for 24 h. Cells were fixed and followed by staining with phalloidin (Actin‐Tracker Green‐488, C2201S, Beyotime) and DAPI (C1005, Beyotime). The images were obtained by scanning confocal fluorescence microscopy.
TEM: DsEVs sample was examined by TEM (Hitachi TEM system, Japan).
NTA: 10 µL DsEVs suspension was loaded into the sample chamber of ZetaVIEW S/N 252 (Particle Metrix GmbH, Germany). Data analysis was performed with software (ZetaView 8.04.02, Germany).
Detection of the loading rate of TAT47‐57‐MBP87‐99A91 into DsEVs: After co‐culturing the FITC‐labeled peptide with DCs for 24 h, DCs were collected and stained with phalloidin (Actin‐Tracker Red‐594, C2205S, Beyotime) and 4′,6‐diamidino‐2‐phenylindole (DAPI, C1005, Beyotime, China). After the isolation of DsEVs.
DsEVs internalized by DCs and CD4+ T cells in vitro: DsEVs were labelled with the Dil (Solarbio life sciences, China) and then co‐cultured with DCs or CD4+ T cells for 24 h. Cells were fixed and followed by staining with phalloidin (Actin‐Tracker Green‐488, C2201S, Beyotime) and DAPI (C1005, Beyotime). The images were obtained by scanning confocal fluorescence microscopy.