Milliplex human cytokine chemokine magnetic bead panel

Cytokine production by infected and uninfected trophoblasts were assessed via luminex assay, using kits to detect 12 human cytokines: IFNγ, IL-10, sCD40L, IL-1RA, IL-2, IL-4, IL-6, IL-8, MCP-1, RANTES, TNFα, and VEGF. Supernatant samples (25 μL) were collected daily and stored at −70 C. Luminex assay was performed using the Milliplex Human Cytokine/Chemokine Magnetic Bead Panel (Millipore) per manufacturer’s instructions. To improve assay sensitivity, samples were incubated with the magnetic beads overnight at 4 °C. Samples were run on a MAGPIX instrument and analyzed with xPONENT software. The results from two donors are shown in Fig. S4.

Cytokine concentrations were measured using a MILLIPLEX Human Cytokine/Chemokine Magnetic Bead Panel (Millipore). For the Tanzanian cohort, a 10-plex (CXCL1, IL-10, IL-1α, IL-1β, IL-8, IP-10, IL-12 p40, IL1-RA, IFN-γ & PDGF-AB/BB) assay was carried out in duplicate on tears diluted 1:10 in assay buffer. For the Gambian cohort, a 3-plex (CXCL1, IL-8 and IP-10) was carried out on undiluted tears in singlicate. Samples from the same participant at different timepoints were assayed on the same plate. Concentrations falling below the range of the standard curve were set to zero (19.2% of measurements in the Tanzanian cohort; 0.1% in the Gambian cohort). One sample in the Tanzanian cohort had insufficient beads and was excluded. Where the median fluorescence intensity was too high to estimate concentration, the concentration was set to that of the highest standard for that cytokine (0.5% of measurements in the Tanzanian cohort; 1.0% in the Gambian cohort). For the Tanzanian cohort the mean of the two replicates was taken. Finally, cytokine concentration was normalised relative to total protein concentration.

Cytokine concentrations were measured using a MILLIPLEX Human Cytokine/Chemokine Magnetic Bead Panel (Millipore). For the Tanzanian cohort, a 10-plex (CXCL1, IL-10, IL-1α, IL-1β, IL-8, IP-10, IL-12 p40, IL1-RA, IFN-γ & PDGF-AB/BB) assay was carried out in duplicate on tears diluted 1:10 in assay buffer. For the Gambian cohort, a 3-plex (CXCL1, IL-8 and IP-10) was carried out on undiluted tears in singlicate. Samples from the same participant at different timepoints were assayed on the same plate. Concentrations falling below the range of the standard curve were set to zero (19.2% of measurements in the Tanzanian cohort; 0.1% in the Gambian cohort). One sample in the Tanzanian cohort had insufficient beads and was excluded. Where the median fluorescence intensity was too high to estimate concentration, the concentration was set to that of the highest standard for that cytokine (0.5% of measurements in the Tanzanian cohort; 1.0% in the Gambian cohort). For the Tanzanian cohort the mean of the two replicates was taken. Finally, cytokine concentration was normalised relative to total protein concentration.

Blood samples were obtained at inclusion and stored at −80 °C in a Biobank at Copenhagen University Hospital in Hvidovre. Creatinine was measured by absorption photometry on a Roche Cobas^® c 8000 701/702 with a module instrument using the Roche Creatinine Plus version 2 IDMS-traceable assay (coefficient of variation 1.5%). NGAL was measured on a Roche Cobas^® c 8000 501/502 with the NGAL Test™ using particle-enhanced turbidimetric immunoassay (PETIA) (Bioporto^®, Hellerup, Denmark) (coefficient of variation 3.7%). suPAR was measured using an enzyme-linked immunosorbent assay (suPARnostic^® Auto Flex ELISA) (ViroGates A/S, Birkerød, Denmark) (coefficient of variation 3%) [43 (link)]. C-reactive protein (CRP) was measured by turbidimetric immunoassay on a Roche Cobas^® 6000 platform in (Roche Diagnostic, Mannheim, Germany) [45 (link)]. Cystatin C was also measured on a Roche Cobas^® c 8000 701/702 with a module instrument using the Roche Cystatin C Tina-quant generation 2 particle-enhanced immunonephelometric assay [45 (link)]. IL-6 and TNFα concentrations were measured on a Luminex^® 200 platform (Luminex, Austin, TX, USA) using the Milliplex Human Cytokine/Chemokine Magnetic Bead Panel (Millipore, Billerica, MA, USA) as described in Klausen et al. 2017 [43 (link)].

Samples were diluted 1:2 in serum matrix for analysis with Milliplex Human Cytokine/Chemokine Magnetic Bead Panel as per manufacturer’s instructions (EMD Millipore Corporation). Concentrations for analytes (EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFNγ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-13, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, TNFβ, and VEGF) were determined for all samples using the Bio-Plex 200 system (BioRad Laboratories, Inc.).

Samples were diluted 1:2 in serum matrix for analysis with Milliplex Human Cytokine/Chemokine Magnetic Bead Panel as per manufacturer’s instructions (EMD Millipore Corporation). Concentrations for analytes (EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFNγ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-13, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, TNFβ, and VEGF) were determined for all samples using the Bio-Plex 200 system (BioRad Laboratories, Inc.).