0.22 μm microcentrifuge filters

Exosomes were isolated from the required cell culture supernatant as described previously (12 (link)). Briefly, the cell culture supernatant was centrifuged (300 × g for 10 min; 2,000 × g for 20 min to eliminate dead cells; and 10,000 × g for 30 min to remove debris) and then pelleted by ultracentrifugation at 100,000 × g for 70 min at 4°C. The pellet was resuspended in 1 ml PBS and re-centrifuged (100,000 × g, 70 min), as aforementioned. The products (UC-Exo) were resuspended in 200 μl PBS and passed through 0.22-μm microcentrifuge filters (Sigma-Aldrich, St. Louis, MO, USA) prior to being stored at −80°C.

Prior to glycan analysis, 0.5 mL of cell culture supernatant was filtered with 0.22 μm microcentrifuge filters (Sigma Aldrich, Dorset, UK) and incubated with Protein A agarose beads (Sigma Aldrich, Dorset, UK) for 2 h at room temperature. After the incubation was complete, the IgG₄ was eluted using 0.2 M Glycine pH 2.5 solution. Following elution, the IgG₄ was transferred to 1× PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) through buffer exchange, using 30 KDa cut-off micro-centrifuge filters (ThermoFisher Scientific, Paisley, UK). For glycan analysis, the C100HT Glycan analysis kit was used (SCIEX, Brea, CA, USA). Briefly, 100 μg IgG₄ was denaturated at 60 °C for 8 min, using the kit’s denaturation solution. Following denaturation, the glycans were digested using 500 units, per sample, of the PNGase F enzyme (New England Biolabs, Hertfordshire, UK) for 20 min and at 60 °C. Released glycans were labelled with APTS-solution at 60 ℃ and for 20 min. Labelled glycans were washed three times with acetonitrile (Sigma Aldrich, Dorset, UK), subsequently eluted in water and loaded to a C100HT Glycan analysis–capillary electrophoresis (SCIEX, Brea, CA, USA) instrument for glycans analysis.