Ht7800 ht7700

After treatment as predesigned, the MCs were immediately soaked in a commercial electron microscope fixing solution at room temperature for 2 h, and then soaked in 1% osmium acid for 2 h at dark-room temperature. After alcohol shaving and dehydration, the cells were permeated and embedded, and then ultrathin slices were made and stained with alcoholic uranyl acetate and alkaline lead citrate. After cleaning and drying, the slices were observed using the transmission electron microscope (TEM, HT7800/HT7700, Hitachi, Tokyo, Japan).
For scanning electron microscope (SEM) observation, the cells were postfixed with 1% of OsO₄ at room temperature in the dark for 2 h. Then they were dehydrated through ethanol series and critical-point dried, mounted on stubs, and sputter-coated with a thin layer of conductive metal, gold, and palladium; finally, the images were observed under SEM (TEM, HT7800/HT7700, Hitachi, Tokyo, Japan).

C. albicans suspensions were adjusted to an initial concentration of 5 × 10⁴ cells/ml, and the suspensions were then transferred to new EP tubes, treated with different concentrations of EVs (0, 120 µg/ml), and cultured in KSFM (37°C, 5% CO₂) for 12 h. Each cell suspension was collected by centrifugation (12000 rpm, 10 min), and the pellet was then suspended and incubated at 4°C in electron microscope fixative for 4h. The samples were washed three times with calcium carbonate buffer solution and treated with 1% agarose solution. The agarose blocks with samples were post fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature while protected from light. After the OsO4 was removed, the tissues were rinsed in 0.1 M PB (pH 7.4) 3 times for 15 min each. Then, the samples were dehydrated with an alcohol and acetone gradient. The cells were then embedded and polymerized. Thin slices (60−80 nm thick) were obtained by using a diamond cutter (Daitome, Ultra 45°) on a Leica superslicer (Leica, Leica UC7). The samples were stained with a uranium acetate-saturated alcohol solution (8 min) and lead citrate (8 min).The cells were then analyzed and photographed using TEM (Hitachi, HT7800/HT7700). The integrated optical density (IOD) and thickness of C. albicans cell wall were quantified and analyzed using Image-Pro Plus image analysis software Version 7.0.1 (Media Cybernetics Inc).

EVs derived from Leuk-1 cells (Leuk-1-EVs) were purified by sequential ultracentrifugation (17 (link)) (Figure 1A). Briefly, Leuk-1 cells were cultured in KSFM medium for 48 h and then centrifuged for 10 minutes at 500 × g to remove cell contamination, 20 minutes at 3,000 × g to remove apoptotic bodies and large cell debris, and 20 minutes at 12,000 × g to remove large microvesicles. After that, EVs were collected by ultracentrifugation in 31 mL ultracentrifugation tubes (#355618, Beckman Coulter, USA) at 110,000 × g for 70 min, washed in PBS, and pelleted again by ultracentrifugation at 110,000 × g in 70 Ti fixed-angle rotors in a Beckman Coulter Optima XPN/XE ultracentrifuge. Finally, the EVs were resuspended in 300 μl of PBS and stored at −80°C.
The ultrastructure and size of EVs were analyzed by transmission electron microscopy (TEM) (Hitachi, HT7800/HT7700, Japan) and subjected to nanoparticle tracking analysis (NTA) with a Zeta View^® system (Particle Metrix, Germany). The protein concentration was measured by BCA assay (Enhanced BCA Protein Assay Kit, Beyotime Institute Biotechnology, China). Western blotting was performed to detect the protein markers of EVs, including CD63 (ab, 59479, Abcam, Cambridge, UK), TSG101 (ab 83, Abcam, Cambridge, UK), and HSP70 (10995-1-AP, Proteintech, Wuhan, China).