Inflammation assay

The BV2 cell line was derived from immortalised murine neonatal microglia and grown in 10% foetal bovine serum and 1% L-Glutamine supplemented Dulbecco’s Modified Eagle’s (DMEM) medium. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, until the cell density reached approximately 1.6 × 10⁶ cell/mL. 200 μL of CSF was diluted in 1 mL of DMEM and added to BV2 microglia cells. Every 24 h the supernatant was removed for analysis and replaced by fresh diluted CSF. The TNF-α concentration in the supernatant was quantified using the Duoset® enzyme-linked immunosorbent assay (ELISA) development system (R&D Systems, Abingdon, Oxfordshire, UK). Three wells for each CSF were used to estimate variation in the experiments. For antibody experiment, CSF and antibody were diluted in DMEM buffer and added to the vesicle containing glass coverslip.