Amicon ultra 3 kda

S. aureus strains were used to inoculate RPMI + 1% CA in glass Erlenmeyer flasks. For aerobic growth, the flask opening was covered lightly with aluminum foil. For hypoxic growth, the flask opening was sealed with a rubber stopper. Cultures were grown for 15 hours. Supernatants were collected after culture centrifugation, and were subsequently filtered through a 0.22 μm filter and concentrated with an Amicon Ultra 3 kDa nominal molecular weight limit centrifugal filter unit (Millipore, Billerica, MA, USA) per the manufacturer’s instructions. Following concentration, supernatants were filter sterilized again and frozen at -80°C until used.

Bacterial pellets were fully grounded in liquid nitrogen. Cells were then dissolved by adding 5–10 mL methanol–water (2:1) solution and packaged into 1.5 mL centrifuge tubes. The mouth of each tube was covered by thin film, which was pierced by a syringe to make small holes. The tube was placed in the diaphragm vacuum pump. The extraction was performed with organic solvent until the samples could be frozen. The samples were combined, with triplicates for each group. NMR detection was performed after freezing and drying.
The samples were treated by Millipore filter membrane (Millipore Amicon^® ULTRA 3 kDa) and ACDSS reagent (DSS standard solution 5 mM). Then, NMR spectrometer (Bruker AV III 600 MHz spectrometer, equipped with a reverse probe) was used for mass spectrometry. The specific parameters were as follows: temperature 298 K, NMR frequency 600.13 MHz, sampling times 128, sample delay 1, frequency domain 65,536, spectral width 12,019.231, number of sampling points 32,768, and pulse sequence noesypr1d/noesygppr1d. Data were analyzed using the Chenomx NMR suit software (version 7.6, Canada).

Cell supernatants were centrifuged at 2000xg for 15 min at 4 °C to exclude cell debris. Supernatants were filtered through 0.22 μm filters (Millipore, USA) and then centrifuged at 110,000xg for 30 min in Amicon Ultra -3 KDa (Millipore, USA). Pellets were resuspended with appropriate PBS and stored at −80 °C. Freshly prepared exosomes were resuspended in 100 μl of 2% PFA, absorbed on Formvar-carbon coated EM grids, washed with PBS, fixed with 1% glutaraldehyde, and washed with water. Thereafter, grid were stained with 4% uranyl-oxalate solution, embedded in 1% methyl cellulose-UA, and observed under electron microscopy at 80 kV. A ZetaView PMX 110 instrument (Particle Metrix, Germany) was used to analyze the distribution of exosome size. Freshly prepared exosome samples were resuspended in PBS and measured according to the manufacturer’s instructions.