Human cytokine 27 plex kit

All CSF samples underwent analysis of cytokines using a custom-made nine-plex kit (Cat No. 12014058, Bio-Rad Laboratories, Hercules, CA) containing eotaxin-1, granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, IL-7, IL-8, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)-1α, and MIP-1β. The nine cytokines were selected based on a screening of a representative set of samples with the human cytokine 27 plex kit (Cat No. M500KCAF0Y, Bio-Rad Laboratories). A 10% bovine serum albumin (Cat No. A5403-50G, lot SLBL9495V, Sigma Aldrich, St. Louis, MO) solution in PBS (pH 7.4, Gibco Cat No. 10010–015, lot 2062123, Thermo Fisher Scientific, Waltham, MA) was added to all CSF samples to a final concentration of 0.5% and vortexed. Then the samples were centrifuged at 10 000×g for 10 min at 4 °C and 50 μL of the supernatant was loaded onto the assay plate. The CSF cytokine levels were measured on a Luminex IS 200 instrument (Bio-Rad). An in-house control was used to observe both intra percentage coefficient of variation and longitudinal (inter) percentage coefficient of variation. Cytokine concentrations below the lower limit of quantification (LLOQ) or above the upper limit of quantification extrapolated by the analysis software were also included in the statistical analysis.