Spark plate reader

Cell viability was assessed in both 2D and 3D cultures using the CellTiter-Glo 3D Cell Viability Assay (Promega) according to the manufacturer's instructions using black opaque-walled multi-well plates suitable for luminescence measurements. In brief, 50 µl medium was removed from each well, and 50 µl CellTiter-Glo 3D was added. All procedures were performed at room temperature. The mixture was pipetted up and down for 30 seconds and assessed microscopically for complete lysis of the spheroid/cells before transferring to a black opaque-walled multi-well plate and incubating on a shaker in the dark for 5 minutes. After a further 25-minute incubation in the dark, luminescence was recorded using a Spark plate reader (Tecan Trading AG, Männedorf, Switzerland).

For studying the cytotoxicity of the different chalcones and the positive control compounds cisplatin and etoposide on HEK293T cells, the Cell Titer^® Blue (CTB) Cell Viability Assay was used (Promega, Mannheim, Germany). A total of 10,000 cells in a 200 µL culture medium were seeded into each well of a 96-well plate. Then, 24 h after seeding, the cells were treated with 0, 1, 2.5, 5, 7.5, 10, 15, 25, 50, or 100 µM of the different compounds (3 wells per compound and concentration) and incubated for an additional 72 h. For measurement of cell viability, 20 µL of CTB reagent were added to each well, and after 5 h at 37 °C, the metabolic activity was quantified by recording the fluorescence signal (λ_Ex 560 nm, λ_Em 590 nm) on a Spark plate reader (Tecan Trading GmbH, Männedorf, Switzerland). Data are shown as mean values ± standard error of the mean (SEM). Statistical significances were calculated by one-way ANOVA (Dunnett’s multiple comparison test), * p < 0.05, ** p < 0.005, *** p < 0.0002, **** p < 0.0001.

The inositol phosphate (IP)-one assay was performed as described previously (Wanka et al., 2018 (link)). In short, confluent HEK293 cells (human embryonic kidney) in 6-well plates were co-transfected with receptor and a chimeric G_α protein (G_αΔ6qi4myr). On the next day the cells were trypsinated, re-seeded in white 384-well plates (Greiner Bio-one) and maintained over night at 37°C, 5% CO₂ and 95% humidity (standard conditions). For measurement of IP production, the IP-one Gq assay kit (Cisbio Bioassay, Codolet, France) was used. The medium was removed and cells stimulated for 1.5 h with 15 μL stimulation solution containing increasing concentrations of NPY or NPY analogs. After cell lysis with lysis buffer supplemented with antibody 1 and antibody 2 according to the manufacturer’s protocol, the emission at 665 nm and 620 nm was measured in a Tecan Spark plate reader (Tecan Group AG, Männedorf, Switzerland).
Using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, United States) the concentration response curves were normalized to the NPY Y₂R curve. The EC₅₀ and E_max values of concentration response-curves were determined by non-linear regression (curve fit). The experiments were conducted in at least two independent experiments.

Antigens used in enzyme-linked immunosorbent assays (ELISAs) were LPS O:9 purified from S. Typhi ZH9 (The Native Antigen Company, Kidlington, UK) and LPS O:2 purified from attenuated S. Paratyphi A (generated by the Wellcome Sanger Institute, Hinxton, UK; purchased from The Native Antigen Company Kidlington, UK). Nunc F96 Maxisorp Immunoplates (ThermoFisher Scientific, Altrincham, UK) were coated with LPS antigens at an appropriate concentration for each batch after pilot experiments determined the optimal range of coating concentrations using control antibodies and checked batch purity. Mouse serum (collected as described above) was added to wells in serial dilutions. Pre-vaccination (d0) samples were pooled across mice to generate a negative assay control. Bound serum antibodies were detected with HRP-tagged secondary goat anti-mouse IgG antibodies (Sigma-Aldrich, Merck Life Science UK Limited, Gillingham, UK) and developed with TMB substrate (Sigma-Aldrich, Merck Life Science UK Limited, Gillingham, UK) for 10 min in the dark. The reaction was stopped with 1 M sulphuric acid, and absorbance was measured at 450 nm within 15 min of adding the Stop Solution with a Spark plate reader (Tecan Group Ltd., Männedorf, Switzerland). End-point titres were calculated by recording the dilution that intersected the curve at OD = 1 for each serum sample.

DNase activity in serum and CSF samples was measured with a DNase I Activity Assay Kit (Fluorometric) (Catalog # K429-100; Biovision Incorporated; Milpitas; California; USA) in Tecan Spark Plate Reader (Tecan Group AG; Männedorf; Schweiz) according to the manufacturers’ instructions with the following changes: the excitation/emission (Ex/EM) was measured at a wavelength of 550/670 nm.

Unless otherwise noted, experiments were performed in white, 384-well microtiter plates (Corning 3572) in 30 μL assay volume. TR-FRET measurements were acquired on a Tecan SPARK plate reader with SPARKCONTROL software version V2.1 (Tecan Group Ltd.), with the following settings: 340/50 nm excitation, 490/10 nm (Tb), and 520/10 nm (AF488) emission, 100 μs delay, 400 μs integration. The 490/10 nm and 520/10 nm emission channels were acquired with a 50% mirror and a dichroic 510 mirror, respectively, using independently optimized detector gain settings unless specified otherwise. The TR-FRET ratio was taken as the 520/490 nm intensity ratio on a per-well basis.