Human xl cytokine premixed magnetic luminex performance assay kit

Chemokines and cytokines were measured by Luminex (Human XL cytokine Premixed Magnetic Luminex Performance Assay Kit (R&D Systems, bio-techne) according to the manufacturer’s instructions. In brief, 50 µl of standard or supernatant inactivated with 1% NP40 for 10 min at 4°C were incubated with antibody-linked beads for 2 h. Then samples were washed three times with wash solution, and incubated for 1 h with biotinylated secondary antibodies. A final incubation of 30 min with streptavidin-PE preceded the acquisition on the Bioplex 200 (Biorad). At least 100 events were acquired for each analyte. Values below the standard curves were replaced by the lowest values of the concentrations measured.

We used MAP technology for multiplexed quantification of cytokines in the CSF and plasma. At every time point, blood was drawn and lumbar puncture was performed. Plasma and aliquots of the CSF sample were centrifuged and the supernatant immediately frozen at −80°C. Samples were stored until use. Assessment of cytokine concentration on CSF and plasma samples was performed using a 27-plex kit (Human XL cytokine Premixed Magnetic Luminex Performance Assay Kit (R&D Systems, bio-techne) according to the manufacturer’s instructions. Analyzed cytokines included IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17, Eotaxin, basic FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP1, MIP-1α, MIP-1ß, PDGF, RANTES, TNF-α and VEGF. In brief, 50 µl of standard, serum or CSF were incubated with antibody-linked beads for 2 h. Samples were then washed three times with wash solution, and incubated for 1 h with biotinylated secondary antibodies. A final incubation of 30 min with streptavidin-PE preceded the acquisition on the Bioplex 200 (Biorad). At least 100 events were acquired for each analyte. Values below the standard curves were replaced by the lowest values of the concentrations measured. In order to enable an optimal comparability between different patients and timepoints, all CSF and plasma samples were measured at the same time on a same multiplex plate.