Bio plex manager 4

To assess cytokine production by isolated B cells, we performed a LUMINEX assay of cell supernatant collected after 20 h of stimulation with TLR9 ligand CpG ODN2216 and after 20 h of unstimulated condition. For the measurements, we used the MILLIPLEX MAP Kit (Merck KGAA, Darmstadt, Germany). Samples were run in triplicates. The protocol was performed according to the manufacturer’s instructions in a 96-well plate with 25 µl of supernatant for cellular protein quantification. Protein quantification was done using the Bio-Plex-System 200 (Bio-Rad Laboratories GmbH, Feldenkirchen, Germany) and the Bio-Plex Manager™ 4.1.1 software (Bio-Rad Laboratories GmbH, Feldenkirchen, Germany), measured in mean fluorescence intensity (MFI). The MFI values were normalized using protein lysate concentrations measured according to the Bradford method.

Sera were separated from clotted blood within 2 h of venesection and stored at −80°C. Serum concentrations of 48 cytokines (see Table S1 in the supplemental material) were assayed using Bio-Plex Pro 21- and 27-plex human cytokine, chemokine, and growth factor fluorescent bead-based assays (Bio-Rad Laboratories). Paired acute- and convalescent-phase samples were assayed together. In brief, antibody-coated fluorescent microspheres were incubated with manufacturer-supplied cytokine standards and study sera, washed, and incubated with detection antibody. Following washing, the microspheres were incubated with streptavidin-conjugated phycoerythrin before data acquisition on a Luminex-100 instrument (Bio-Rad Laboratories) using Bio-Plex Manager 4.1.1 software (Bio-Rad). Cytokine measurements below the detection limit of the assay were assigned values of the lower detection limit for each cytokine and were included in the analysis. At least one multiplexed assay failed in the case of five samples (3 acute, 2 convalescent), and these were excluded from analysis.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) were measured with Luminex^®, according to the manufacturers’ protocol using customized Procartaplex^TM Immunoassay Kits (Life Technologies, Merelbeke, Belgium). In short, 96-well plates were loaded with antigen-specific capture antibody-coated magnetic beads before samples; standards and blanks were added and incubated for two hours. Subsequently, biotinylated detection antibodies were added. The antibody/antigen complex was visualized using streptavidin-conjugated R-phycoerythrin. A magnetic plate washer was used for washing steps throughout the protocol. Read-out was performed with Bio-plex 200 system of Bio-Rad (Hercules, CA, USA). Concentrations of the proteins were determined using five parameter log curves generated by the Bio-Plex Manager 4.1.1 software (Bio-Rad, Hercules, CA, USA).