Bio plex 200 station

A panel of cyto- and chemokines known to be involved in the pathophysiology of bacterial meningitis was selected to document the inflammatory response in the present infant rat model of PM [49] (link): tumor necrosis factor (TNF-α), interleukin-6 (IL-6), IL-1β, IL-10, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1 α (MIP-1α), interferon gamma (IFN-γ) [50] (link). The concentration of these analytes was determined in the CSF using microsphere-based multiplex assays (MILLIPLEX MAP Kit, Rat Cytokine/Chemokine Magnetic Bead Panel, Cat. #RECYTMAG-65K; Millipore Corporation, Billerica, MA). 5 µl of CSF supernatant were diluted to a final volume of 25 µl using the provided assay buffer. A minimum of 50 beads per analyte was measured using a Bio-Plex 200 station (Bio-Rad Laboratories, Hercules, CA). Calibration curves from recombinant standards were calculated with Bio-Plex Manager software version 4.1.1 using a five-parameter logistic curve fitting. If the sample concentration was below detection limit, the value of the detection limit as provided by the manufacturer and multiplied by the dilution factor used for statistical analysis, i.e. TNF-α 9.5 pg/mL, IL-6 153.5 pg/mL, IL-1β 14 pg/mL, IL-10 13.5 pg/mL, MCP-1 45 pg/mL, MIP-1α 4.0 pg/mL, IFN-γ 31 pg/mL.

Cyto-/chemokines and MMP-9 in cerebella homogenates were quantified by using microsphere-based multiplex assays (Milliplex MAP mouse cytokine/chemokine multiplex assay and Milliplex MAP mouse CVD panel 1 multiplex assay, Merck Millipore, Billerica, MA, USA). Cerebella were homogenised with a glass Dounce homogeniser in 1:7 (w/v) extraction buffer containing 0.1% Triton X-100 and a protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland) in PBS. One hundred μg (for cyto-/chemokines) or 1 μg (for MMP-9) of homogenates were tested in duplicate. A minimum of 50 beads per analyte was measured using a Bio-Plex 200 station (Bio-Rad Laboratories, Hercules, CA, USA). Calibration curves from recombinant standards were calculated with the Bio-Plex Manager software version 4.1.1 using a five-parameter logistic curve fitting.

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.

Cytokines and chemokines (IL-1β, IL-6, TNF-α, IL-10, RANTES and MIP-1α) were assessed using magnetic multiplex assay (Rat Cytokine/Chemokine MagneticPanel, Milliplex® Map Kit, Merck) on a Bio-Plex 200 station (Bio-Rad Laboratories) as previously described^{19 (link),30 (link)}. If the concentration of the sample was below the detection limit, a value corresponding to the detection limit provided by the manufacturer was used, considering the dilution factor. Detection limit for the samples were TNF-α 1.9 pg/mL, IL-1β 2.8 pg/mL, IL-10 2.7 pg/mL, IL-6 30.7 pg/mL, RANTES 1.3 pg/mL and MIP-1α 0.8 pg/mL.

A panel of PM-associated cytokines (TNF-α, IL-6, IL-1β, IFN-γ, and IL-10) was measured in the CSF at 18, 24 and 42 hpi by using a 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 previously described (8 (link), 37 (link)). Five microliters CSF were diluted to a final volume of 50 μl and at least 50 beads per analyte were measured. Calibration curves from recombinant standards were calculated with Bio-Plex Man-ager software (version 4.1.1) using a five-parameter logistic curve fitting. For samples below the detection limit, the value of detection limit provided by the manufacturer (TNF-α, 22.1 pg/ml; IL-6, 56.0 pg/ml; IL-1β, 26.7 pg/ml; IL-10, 18.6 pg/ml; IFN-γ, 70.5 pg/ml) was multiplied by the dilution factor.

Five cytokines (IL-6, IFN-γ, MCP-1, IP-10, and RANTES) were assessed in the CSF and in the blood serum using a magnetic multiplex assay (MILLIPLEX MAP Rat Cytokine/Chemokine Magnetic Bead Panel–Immunology Multiplex Assay, Millipore) on a Bio-Plex 200 station (Bio-Rad Laboratories). For the three timepoints (day 4, day 9, and day 21 pi) 10 μL of CSF was diluted to a 50 ul final volume. For statistical purposes, values for the samples below the detection limit were calculated using the detection limit provided by the manufacturer multiplied by the dilution factor (IL-6 30.7 pg/mL, IFN-γ 6.2 pg/mL, MCP-1 9.0 pg/mL, IP-10 1.4 pg/mL, RANTES 1.3 pg/mL).

Cytokines known to be upregulated during PM (IL-1β, IL-6, TNF-α, IL-10, and IFN-γ) 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 [35 (link), 60 (link)]. Five microliter of CSF harvested at 18, 24, and 42 hpi were diluted to a final volume of 50 μl. For in vitro samples, 50 μl of cell culture medium was used undiluted. 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 detection limit provided by the manufacturer was used, considering the dilution factor. 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, 11.5 pg/ml for TNF-α, and 70.9 pg/ml for IFN-γ.