Cytotox 96 non radioactivity cytotoxicity assay

IFNβ concentrations in culture supernatants were measured by ELISA according to the manufacturer’s instructions (MSD, Gaithersberg, USA). Culture supernatants were analysed for IL-10 by Luminex assay as per manufacturer’s instructions (Bio-Rad). LDH release was measured using CytoTox 96® Non-Radioactivity Cytotoxicity Assay according to the manufacturer’s instructions (Promega, Southampton, UK). Release of viral hemagglutinin was measured using a dot blot assay on nitrocellulose membrane detected using a rabbit polyclonal anti-influenza serum. The bound rabbit antibodies were detected using the Bio-Rad anti-Rabbit HRP detection system according to the manufacturer’s instructions.

Peripheral blood samples were collected on days -2 (pre-Vac; 2 days before the first vaccination) and 60 (post-Vac: 18 days after the fourth vaccination). Approximately 100 μl of whole blood was collected from the tail vein of vaccinated mice and kept at room temperature for 30 min to enable clotting. After centrifugation at 10,000 ×g at room temperature for 10 min, the serum in the upper layer was collected and kept at -20°C until use. The collected sera were diluted and subjected to the measurement of peripheral αSyn by using the Alpha-synuclein SimpleStep ELISA kit (#ab282865, Abcam), 20 inflammation-related cytokines by using the Quantibody® Mouse Cytokine Array 1 (#QAM-CYT-1, RayBiotech), and lactate dehydrogenase (LDH) by using the CytoTox 96® Non-radioactivity Cytotoxicity Assay (#G1780, Promega), according to manufacturer’s instructions.

IFNβ concentrations in culture supernatants were measured by ELISA according to the manufacturer’s instructions (MSD, Gaithersberg, USA). Culture supernatants were analysed by Luminex assay as per manufacturer’s instructions (Bio-Rad). LDH release was measured using CytoTox 96 Non-Radioactivity Cytotoxicity Assay according to the manufacturer’s instructions (Promega, Southampton, UK). Release of viral hemagglutinin was measured using a dot blot assay on nitrocellulose membrane detected using a rabbit polyclonal anti-influenza serum. The bound rabbit antibodies were detected using the Bio-Rad anti-Rabbit HRP detection system according to the manufacturer’s instructions.