Infinite f200 pro instrument

The DELFIA^® immunoassay is a time-resolved fluorescence method that can be used to study antibody binding to solid-phase proteins or peptides. A set of sera, comprising 9 T. cruzi-infected patient samples and 15 HD samples, was analyzed against recombinant TcSMP. Purified recombinant TcSMP (20 μg/mL) in 1X PBS was used to coat DELFIA plates (PerkinElmer). The serum assay procedure was automatically performed by the Freedom-EVO Liquid Hander (Tecan, Männedorf, Switzerland), as previously described [36 (link)]. In brief, plates were blocked for 1 h at 37 °C with a blocking reagent (Perkin Elmer Italia S.r.l., Milano, Italy). Serum samples were diluted 1:300 in 0.1% (v/v) TPBS containing 1% (w/v) BSA and incubated for 1 h at 37 °C. Plates were then washed four times with washing buffer (PerkinElmer) and incubated for 30 min at RT in the dark with Europium-labeled α-Human IgG serum (1:500 in diluting buffer, PerkinElmer). After extensive washing by Hydrospeed™ (Tecan), plates were left at RT for 10 min and then read on an Infinite F200 PRO instrument (Tecan). Fluorescence intensity values, higher than the mean of HD plus one standard deviation, were considered as positive. DELFIA results were analyzed using Student’s t-test for the MFI, as well as the two-sided Fisher’s exact test to concern the recognition frequency, using the GraphPad software 9.2.0.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined with a Seahorse XFe96 Analyzer (Seahorse Bioscience). To this end, 40,000 wild-type or mutant ESCs were seeded into 7–8 wells of a gelatine-coated 96-well analyzer plate (Seahorse Bioscience) and incubated overnight under standard conditions. For determination of OCR, oligomycin (2 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (0.6 µM), and rotenone/antimycin A (0.5 μM) were injected according to the XF Cell Mito Stress Test Kit (Seahorse Bioscience). Glycolytic function was determined using the XF Glycolysis Stress Test Kit (Seahorse Bioscience). Glucose (10 mM), oligomycin (2 µM), and 2-deoxy-glucose (2-DG) (50 mM) were injected according to the manufacturer’s protocol. The obtained OCR (respiration) and ECAR (glycolysis) data were normalized against cell numbers in each well and analyzed using the Wave software as well as the XF Report Generator (Seahorse Bioscience). Cell numbers were determined by Hoechst 33342 (Thermo Scientific) staining combined with fluorescence measurement using Infinite F200 PRO instrument (Tecan).

In the Liver-BIBLE 2020 cohort, circulating levels of IL32 were quantified using Human IL32 DuoSet ELISA kit (DY3040, R&D Systems, Minneapolis, USA) following the manufacturer’s instructions. The assay is designed to detect IL32α, IL32β and IL32γ with a detection range of 78.5–5000 pg/mL. Plasma Samples were diluted 1:2-1:20 in PBS and were measured in duplicate. Optical density was measured at 450nm using TECAN infinite F200 PRO instrument (Männedorf, Switzerland). The minimal detectable concentration above blank was 10 pg/mL, and all samples measured at lower levels were set at 10 pg/mL. The intra-assay coefficients of variation (CV) were 3.9 ± 5.0%. PLA2G2A was measured in cell culture supernatant of primary human hepatocyte spheroids by Human PLA2G2A ELISA kit (Thermo fisher scientific) following the manufacturer’s instructions. PLA2G2A encodes for secretory phospholipase A2, an extracellular enzyme. Briefly, 100μL of media was collected from 10 wells that contain spheroids, and pooled together for each technical replicate. Samples were measured in triplicates. Undiluted media was measured, and the detectable concentration range was found to be high. Thus, the samples were diluted two times with PBS and the kit-provided-standards were used to plot the standard curve every time the ELISA was performed.

For the experimental verification of the antigens selected, DELFIA^® assay, a time-resolved fluorescence method, was used as described previously [31 (link)]. In brief, 20 μg/ml purified recombinant proteins (in 6M Urea) were coated in a 384-well format plates in duplicate with the Fredom-Evo Liquid Handling (Tecan). The plates were blocked with a blocking reagent (PerkinElmer) than diluted sera was incubated for 1 hour at 37°C. The plates were then washed 5 times with washing buffer (PerkinElmer) and probed for 30 min at room temperature in the dark with Europium-labeled α-human IgG serum (1:500 in diluting buffer, PerkinElmer). After washing, using Hydrospeed™ (TECAN), plates were left at room temperature for 10 min and finally read by an Infinite F200 PRO instrument (Tecan). Fluorescence intensity values higher than the mean of HD plus 1 SEM were considered as positive.
DELFIA^® results were analyzed using the two-tailed X² test, the Student’s t test, the Fisher’s exact tests, or the analysis of variance test using either TIGR Multiexperiment Viewer and GraphPad software.
The Epicalc package was used to obtain the Receiver Operating Characteristic (ROC) curves of the models and the area under the curve (AUC) values.
STRING software (http://string-db.org) [38 (link)] was used to analyze the biological network of UNQ9419 and CHAD.

For a fluorescence polarization assay, a protein sample (cjRecR, cjRecO, and their mixture) at various concentrations up to 100 μM was incubated with a fluorescein-probed 40-mer ssDNA (5′-TTATAGGCATATAGGAGTAATTTTCTTGGGCTATGCAGTA-3′; 0.8 nM). The fluorescence polarization level of the protein–ssDNA solution was determined using an Infinite F200 PRO instrument (Tecan, Männedorf, Switzerland). The dissociation equilibrium constant (K_d) for the cjRecR–ssDNA interaction was estimated using a one-site binding model with the Prism 5 software (GraphPad, San Diego, CA, USA).

An FP assay was performed to determine the ssDNA-binding affinities of cjRecO and cjSSB. A fluorescein-labeled 40-mer ssDNA (5′-TTATAGGCATATAGGAGTAATTTTCTTGGGCTATGCAGTA-3′; 0.8 nM) was incubated with each protein at various concentrations in 20 mM Tris, pH 8.0, 30 mM NaCl, and 5 mM βME, and then the FP of the fluorescein-labeled ssDNA was measured using an Infinite F200 PRO instrument (Tecan, Männedorf, Switzerland). A K_d value was derived with the Prism software (version 5.01, GraphPad, San Diego, CA, USA) using a one-site binding model.