Nanophotometer n120

The anti-GH capture antibody was biotinylated using the protein biotin labeling kit. A volume of 500 µL of the antibody (1 mg/mL in PBS, pH 7.2, carrier free) was incubated with 22.0 µL freshly prepared biotin-7-NHS labeling solution (3 mM in DMSO) for 120 min at room temperature and 600 rpm and protected from light. After biotinylation, the remaining non-reacted biotin-7-NHS was removed using a Sephadex G-25 gel filtration column. The concentration of the biotinylated capture antibody was determined using the Implen NanoPhotometer® N120 (München, Germany) at 280 nm against a corresponding blank solution and found to be 173 µg/mL. The biotinylated antibody solution was divided into 0.1-mL aliquots in Eppendorf Protein Lobind tubes and stored at − 80 °C.

Total genomic DNA was extracted from the parasite biological materials from each cyst belonging to the 13 human patients (HCE1-HCE13), detected following the isolation, respectively, of hydatid fluid and germinal layer. In detail, protoscoleces were removed by gently scraping the germinal layer placed in a Petri dish together with the hydatid fluid and then washed twice in phosphate-buffered saline (PBS) by a centrifuge step (10 min at 1000× g). Next, the supernatant was discarded, and 25 mg of the pellet were aliquoted and stored at −80 °C until the DNA extraction step. Conversely, negative control was obtained from a DNA sample extracted from a healthy human lymph node. DNA extraction was performed using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total genomic DNAs were quantified by a NanoPhotometer^® N120 (Implen GmbH, Munich, Germany), according to the manufacturer’s instructions. Moreover, agarose gel electrophoresis was employed to check the presence and the integrity of the extracted total genomic DNAs. A 0.5% gel was prepared by melting the agarose powder in TAE buffer 1X; then, ethidium bromide (EtBr) was added to a final concentration of 0.5 μg/mL. DNA was loaded into the gel wells along with a molecular weight ladder. Finally, the electrophoretic run was performed at 150 V and 400 mA for about 1 hour.

Total RNA was isolated from treated THP-1-derived macrophages using Trizol reagent and chloroform extraction technique following the manufacturer’s instructions. RNA concentrations were determined using the NanoPhotometer^® N120 (Implen; München, Germany). First, 1 µg of total RNA was reverse transcribed to cDNA using a Maxima First strand cDNA synthesis kit. Quantitative analysis was achieved using real-time quantitative PCR (RT-qPCR), with each cDNA sample performed in triplicate. qPCR was realized using the BioRad CFX384 Touch Real-Time PCR Detection system and iTaq SYBRgreen qPCR mix. Table 1 lists the specific primers used for qPCR, Nrf2, HO-1, NQO1, IL-6, IL-1β, TNF-α, and 18S rRNA, and synthetized by Eurogentec (Seraing, Belgium). The cycling parameters for qPCR were 95 °C for 3 min, 40 cycles of 95 °C for 5 s and 60 °C for 20 s, with a melting curve from 65 °C to 95 °C. The cycle threshold (Ct) values of each target genes were first normalized to that of the reference gene 18S rRNA (ΔCt) then the final values (ΔΔCt values) were expressed as folds of control. Data were analyzed on the BioRad CFX manager 3.1 using the ΔΔCt method.

Total RNA was extracted using TRIzol method, and its concentration was tested by NanoPhotometer N120 (IMPLEN, Germany). Real-time PCR was performed as described previously 33 , 34 (link). The primers are listed in the supplementary Table S3. RNA sequencing was performed by BMK (www.biomarker.com.cn). Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample, sequenced using a NovaSeq 6000 (Illumina) according to the manufacturer's instructions.