Tecnai g2 spirit twin tem

Spleens were extracted from WT mice. Following single-cell isolation, splenocytes were fixed with 2.5% glutaraldehyde in 0.1 M DPBS for 2 h at 4 °C and then postfixed for 2 h in 2% osmium tetroxide. In the process of gradually increasing the concentration of ethanol and propylene oxide, the fixed samples were dehydrated and finally embedded in EMbed-812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Polymerization was performed at 60 °C for 24 h, and an ultramicrotome (Leica, Wetzlar, Germany) was used to prepare ultrathin (100 nm) sections.
For immunogold labeling, prepared sections were reacted with etching solutions for 1 h and then blocked with Tris-buffered saline containing 4% goat serum, 1% bovine serum albumin, and 0.1% Tween-20. The sections were incubated with a 1:100 dilution of IFT20 antibody overnight at 4 °C. The next day, the sections were stained with a gold-conjugated goat anti-rabbit IgG secondary antibody (AURION, Wageningen, Netherlands) at a 1:100 dilution for 2 h at room temperature. Finally, the sections were sequentially dyed with 2.5% uranyl acetate for 7 min and lead citrate for 3 min. Images were captured with a Tecnai G2 spirit TWIN TEM (FEI Company, Hillsboro, OR, USA) at an acceleration voltage of 120 kV. The antibodies used for immunogold labeling are listed in Supplementary Table 4.

Cryo-TEM images were obtained using a BIO-TEM installed at the Korea Basic Science Institute (Osong, Korea). The isolated ASC-exosomes were applied to Quantifoil grids (Electron Microscopy Sciences, Hatfield, PA, USA) and subsequently blotted using Vitrobot Mark IV (FEI, Hillsboro, OR, USA) with the following settings: 100% humidity, 4 °C, blot time of 5 s, blot force of 5, blot total of 1, wait time of 5 s, and drain time of 0 s. For maintenance of vitrified sample grids at a temperature of around −178 °C within the TEM, a side entry Gatan 626 cryo-holder (Gatan, Pleasanton, CA, USA) was used. The grids were then examined with a Tecnai G2 Spirit Twin TEM equipped with an anti-contaminator (FEI Company, Hillsboro, OR, USA). A 4K × 4K, Ultrascan 4000 CCD camera (Gatan) was used for image recording. A low-dose method (exposures at 1000 electrons per nm²/s) was used for cryo-TEM.

Following the synthesis, the nanoliposomes were diluted 1:5000 and 1:10 for nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS), respectively. NTA measurements were recorded and analyzed using the Nanosight NS300 (Malvern, UK); DLS and Zeta potential measurements were performed using the Brookhaven Zeta-PALS light-scattering analyzer (New York, NY, USA). Fourier-transform infrared spectroscopy was performed using the Perkin Elmer FTIR Spectrum II (Waltham, MA, USA) on lyophilized samples. Absorbance measurements were performed in 96-well plates at various dilutions using the Spectramax i3 (Molecular Devices, San Jose, CA, USA). For transmission electron microscopy, immediately after synthesis, liposomes were drop cast on a carbon-copper grid and stained with uranyl acetate for contrast. The imaging was performed using the FEI Tecnai G2 Spirit Twin TEM (Hillsboro, OR, USA) at a voltage of 120 kV.