Bio plex 200 analyser

Cell culture supernatants were assayed by Human Magnetic Luminex Screening Assay ELISA (R&D Systems, Minneapolis, MN, USA) to measure the concentration of inflammatory factors monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, regulated upon activation, normal T cell expressed and presumably secreted (RANTES), and intercellular adhesion molecule (ICAM)-1. EA.hy926 cells were incubated with the FAs in 96 well plates (1 × 10⁴ cells/100 µL per well) for 48 h and then incubated with TNF-α for a further 24 h. Before the supernatants of each well were collected and stored at −80 °C until analysis, the cells were checked under the microscope. Assays were conducted in accordance with the instructions from the manufacturer. Plates were analysed on a calibrated Bio-Plex 200 analyser using Bio-Plex software (version 6.1, Bio-Rad Laboratories Inc., Berkeley, CA). Lower limits of detection (pg/mL) were IL-6, 1.7; IL-8, 1.8; MCP-1, 9.9; RANTES, 1.8; ICAM-1, 87.9. Due to differences in the ranges of fluorescence values among experiments, the results are presented as % of control.

The mouse IFNα platinum ELISA kit (normal sensitivity; Affymetrix eBioscience, Vienna, Austria) or the Verikine-HS mouse IFNα All subtype ELISA kit (high sensitivity; Pbl Assay Science, Piscataway, NJ, USA) was used to measure serum IFNα concentrations from LCMV infected mice. The Verikine mouse IFNβ ELISA kit (normal sensitivity; Pbl Assay Science) or the Verikine-HS mouse IFNβ ELISA kit (high sensitivity; Pbl Assay Science) was used to measure serum IFNβ concentrations from LCMV infected mice. These are standard ELISA kits that were performed according to manufacturer’s instructions. Alternatively, mouse IFNα and IFNβ were also measured using the mouse interferon dual plex (#EPX02A-22187-901, Thermofisher Scientific). A cytokine multiplex array was also performed using the ProcartaPlex™ 26-Plex Panel 1 (EPX01A-26088-901), according to manufacturer’s instructions (ProcartaPlex, Thermofisher Scientific). Plates were read using a Bioplex 200 analyser (Biorad, Hercules, CA, USA).

Cell culture supernatants were assayed using the Human Magnetic Luminex Screening Assay ELISA (R&D Systems, Minneapolis, MN, USA) to measure the concentrations of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, regulated upon activation, normal T cell expression and presumably secreted (RANTES) and intercellular adhesion molecule (ICAM)-1. The EA.hy926 cells were incubated with the FAs in 96-well flat-bottomed plates (Corning Corning, NY, USA) (1 × 10⁴ cells/100 µL per well) for 48 h and then without FAs for 24 h. Supernatants were collected and stored at −80 °C until analysis. Assays were conducted in accordance with the manufacturer’s instructions. Plates were analysed on a calibrated Bio-Plex 200 analyser using Bio-Plex software (version 6.1, Bio-Rad Laboratories Inc., Berkeley, CA, USA). Lower limits of detection (pg/mL) were IL-6, 1.7; IL-8, 1.8; MCP-1, 9.9; RANTES, 1.8; ICAM-1, 87.9. Due to the differences in the ranges of fluorescence values among the experiments the results are presented as % of control.

Brain and plasma samples were collected at 6 h, 1 day, 3 days, 7 days and 14 days after HI. Briefly, mice were deeply anaesthetised, blood was collected from the heart’s right ventricle and transferred to EDTA-coated tubes and set aside for further processing, animals were then transcardially perfused with ice-cold 0.9% saline and brains were rapidly removed and frozen on dry ice. Plasma samples were isolated via centrifugation (10 min, 1000×g, 4 °C) and frozen on dry ice prior to storage at − 80 °C.
Brain lysates were prepared through mechanical dissociation of tissue samples in 600 μl of lysis buffer (10 mM EDTA, 1% Triton-X-100, 1% Protein Inhibitor Cocktail [Sigma-Aldrich#8340] in RNase-free PBS) followed by sonication and centrifugation (4500×g, 5 min, 4 °C). Protein concentration was assessed using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer’s protocol, and the final concentration of the samples was adjusted to 1 mg/ml using the lysis buffer. Preparations were carried out at 4 °C, and samples were stored at − 80 °C.
Cytokine concentration in brain lysates and plasma samples were assessed using a Bio-Plex Pro Mouse Cytokine Standard 23-Plex (Bio-Rad) kit in accordance with manufacturers’ instructions on a Bio-Plex 200 analyser. For brain samples, the results were normalised to the brain protein concentration.

Cytokines were quantified in EDTA-treated plasma using the NHP cytokine Milliplex (Merck Millipore, Darmstadt, Germany) for IL-6, TNF-α, IL-1β and MCP-1 (also known as CCL2); the NHP metabolic Milliplex panel for leptin and insulin; and the Human Adipokine Milliplex for adiponectin. Analysis was performed on a Bioplex 200 analyser (Bio-Rad) according to manufacturer’s instructions.

A customised Luminex multiplex immunoassay was used to assess IgG antibody responses against the major capsid protein VP1 of LIPyV and 13 other HPyVs, namely BKPyV, JCPyV, Karolinska Institute polyomavirus (KIPyV), Washington University polyomavirus, MCPyV, Human polyomavirus 6 (HPyV6), Human polyomavirus 7 (HPyV7), TSPyV, Human polyomavirus 9, Malawi polyomavirus (MWPyV), Saint Louis polyomavirus (STLPyV), HPyV12, and NJPyV (Table 1). This assay was previously described in detail [27 (link)]. Briefly, VP1 fusion proteins were expressed in E. coli and coupled to uniquely coloured, magnetic fluorescent beads (Bio-Rad Laboratories, Hercules, CA, USA). Biotinylated goat-α-cat IgG, rabbit-α-dog IgG, and goat-α-human IgG (H+L) (Jackson ImmunoResearch, Cambridgeshire, United Kingdom, dilution 1:1000) were used as secondary conjugate antibodies that were detected with streptavidin-R-phycoerythrin (SAPE) (1:1000). Antibody responses were measured in a Bio-Plex 200 analyser (Bio-Rad Laboratories, Hercules, CA, USA) and analysed using Bio-Plex Manager 6.1 software. Specific antibody responses were calculated by subtracting from each sample the median fluorescence intensity (MFI) values of a blank sample (no serum added) and of beads coupled to SV40 small t protein as a background measurement. An arbitrary cutoff for seropositivity was set at 1500 MFI.