Exosome depleted fbs

1,2-dipalmitoyl-sn-glycero-3-phospocholine (DPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids Inc. (Birmingham, AL, USA). Lissamine Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3 phosphoethanolamine (rhodamine-DHPE) was provided by ThermoFisher Scientific Co., Ltd. (USA). Disodium fluorescein was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipopolysaccharide (LPS) was provided by InvivoGen (San Diego, CA, USA), and Interferon gamma (INF-γ) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The cell line J774A.1 was obtained from American Type Culture Collection (ATCC, USA). Fetal bovine serum (FBS) and exosome-depleted FBS were provided by Thermo Scientific (Paisley, UK). All the other reagents used in the experiments were of analytical grade (> 98%).

Twenty-four hours after transfection, cells were washed in PBS twice, and then 2.5 × 10⁶ cells of transfected cells were re-plated on 10-cm dish with 10 ml of media supplied with 10% exosome-depleted FBS (Thermofisher Scientific, Waltham, MA, USA) and anti-biotic anti-mycotic. After 72 h, culture media was collected, and centrifuged at 2000g for 30 min at 4 ℃ to eliminate cells and debris. The supernatant was incubated with total exosome isolation reagent overnight at 4 ℃ (Thermofisher Scientific, Waltham, MA, USA). The mixture was centrifuged at 10,000g for 60 min at 4 ℃. The pellets contained extracellular vesicles (EVs), including apoptotic bodies, were used for Western blotting as described in “Supplementary Methods”. Immunofluorescent labeling, confocal imaging, and protein expression quantification are also detailed in “Supplementary Methods”.

Exosomes were prepared using a polyethylene glycol (PEG)-based method [18 (link)]. To isolate exosomes from the conditioned medium of macrophages, cells were cultured in DMEM supplemented with 10% exosome-depleted FBS (ThermoFisher) for 24 h and exosome-containing supernatant was collected. Cell debris was removed by centrifuge at 1500 g for 10 min. Ten mililiter of exosome-containing supernatant was mixed with 5 mL 34% PEG 6000 (Sigma) solution. To isolate exosomes from serum, 50 μL serum was diluted in 150 μL PBS followed by mixing with 100 μL 34% PEG solution. After intensive mixing, the mixture was incubated overnight (at least 14 h) at 4 °C followed by centrifugation at 3000 g for 1 h. The supernatant was completely removed and the remaining pellet at the bottom of the tube was resuspended in RIPA buffer for 3 min on ice to prepare exosomal lysate or resuspended in PBS for following experiments. The quality of exosomes was confirmed by dynamic light scattering using a particle and molecule size analyzer (ZetasizerNano ZS, Malvern Instruments) according to the manufacturer’s instruction. The presence of exosome markers, including HSP70, CD63, Alix, and the absence of calnexin was confirmed by western blot.

Forty-eight hours before isolation, 1 × 10⁷ HeLa cells were plated in 150-mm tissue culture dishes. Culture medium was removed and replaced with 20 ml of DMEM with exosome-depleted FBS (Thermo Fisher Scientific, catalog no. A2720801) and incubated for 48 hours. Then, cells were incubated with DMEM in the presence or absence of exosome-depleted FBS for an additional 16 hours. Conditioned medium was collected, transferred to 50-ml tubes, and centrifuged for 20 min at 2000g at 4°C. Supernatants were transferred to a fresh 50-ml tube and centrifuged for 30 min at 10,000g at 4°C. Supernatants were ultracentrifuged for 90 min at 35,000 rpm at 4°C in a Ti-90 rotor to pellet ECVs. To fix the samples for EM, 16% paraformaldehyde (PFA) was added to a final concentration of 2% PFA. When assessing by Western blot, normalization was performed by controlling for the number of cells seeded onto the plate.

Vesicle binding to C. albicans was analyzed using a modified protocol by Zhao et al. (67 (link)). Extracellular vesicles from PBMCs (1 × 10⁶) were stained with Vybrant DiD cell-labeling solution (2 μM) (Thermo Fisher Scientific) for 5 min at room temperature (RT). For blocking assays, EVs were incubated with monoclonal CR1 or CR3 antibody (each 5 μg/ml) (BioLegend) in exosome-depleted fetal bovine serum (FBS) (1%, diluted with 1× PBS) for 30 min at room temperature. The labeling or blocking reaction was stopped with exosome-depleted FBS (System Biosciences) (1% diluted with 1× PBS) and vesicles isolated using the total exosome isolation solution (Thermo Fisher Scientific) and centrifugation at 10,000 × g for 1 h. The washing step was repeated twice and the EV pellet resuspended in exosome-depleted FBS (1% diluted with 1× PBS). Opsonized C. albicans cells (5 × 10⁵) were incubated with EVs for 30 min with shaking at 37°C. In parallel, untreated C. albicans cells were stained with Vybrant DiD cell-labeling solution (2 μM). Yeast cells were washed three times with 1× PBS and binding of vesicles was recorded by flow cytometry using BD Accuri C6 plus (Becton Dickinson).