Cytokines known to be upregulated during L. monocytogenes meningitis (IL-1β, IL-6, IL-10, IL-18, TNF-α, and VEGF) (Koopmans et al., 2014 (link), 2018 (link)) were assessed using magnetic multiplex assay (Rat Magnetic Luminex® Assay, Rat Premixed Multi-Analyte Kit, R&D Systems, Bio-Techne) on a Bio-Plex 200 station (Bio-Rad Laboratories) as described previously (Perny et al., 2016 (link); Muri et al., 2018 (link)). Cerebellum homogenates were centrifuged (16,000 × g, 10 min, 4°C) and protein concentration determined using Pierce™ BCA Protein Assay kit (ThermoFischer Scientific). 100–150 μg proteins were diluted to a final volume of 50 μl. For each sample, a minimum of 50 beads was measured. If the concentration of the sample was below the detection limit, a value corresponding to the lower limit of detection provided by the manufacturer was used. The detection limits for undiluted samples were 2.93 pg/ml for IL-1β, 23.2 pg/ml for IL-6, 8.95 pg/ml for IL-10, 3.32 pg/ml for IL-18,11.5 pg/ml for TNF-α, and 15.6 pg/ml for VEGF.
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