Amicon ultra 4 ml

The purified exosomes were stained with a PKH26 red fluorescent cell linker kit (Sigma-Aldrich). After incubation with PKH26, exosomes were washed using 100 KDa filters (Amicon Ultra-4 mL, Millipore, Darmstadt, Germany) to eradicate redundant lipid dye. For the cellular uptake of exosome analysis, naïve CD4⁺ T cells were preactivated under stimulation with anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) mAbs for 24 hours and cocultured with labelled exosomes for another 24 hours. The results were detected under a confocal microscope.

For comparative secretome analysis, 20 mL of culture supernatant was grown in brain heart infusion (BHI) broth to logarithmic stage (OD_600nm = 0.6), and concentrated to a 500 μL vol. (40-fold concentration) using AmiconUltra-4 mL (3 kDa m.w. cutoff) (Millipore, MA, United States), the tested loading samples were ready to be analyzed for DIA mass spectrometry by the Kazusa DNA Research Institute (Kisarazu, Japan). Briefly, 20 μg of secreted total protein was reduced by dithiothreitol, followed by iodoacetamide alkylation at cysteine residues, and digested by Lys-C protease and trypsin. The digested peptides were purified, followed by nanoLC-MS analysis with a nanoLC: UltiMate 3000 RSLCnano LC System (Thermo Fisher Scientific, MA United States) and MS: Q Exactive HF -X (Thermo Fisher Scientific). The detected MS peaks were analyzed by Scaffold DIA Proteome Software using coding sequences in KG-18 as references.