MTT assay

Some 2.5 × 10⁴ THP1 cells were seeded in a 96-well plate (Falcon) and then differentiated into adherent macrophages. The cells should be 80%–100% confluent before starting the assay. MTT reagent stock (Life Technologies, catalogue no. M6494; 5 mg ml⁻¹ in PBS) was diluted 1:10 in 37 °C RPMI complex medium. After removal of supernatant, 50 µl of the prepared dilution was added to the cells and incubated at 37 °C for 20–60 min while periodically observing the colour in the cells. The reagent was removed when staining was completed, and 50 µl of dimethylsulfoxide (Merck) was added. After mixing on a shaker for 5–15 min (until the solution was homogeneous), the colour change was quantified by enzyme-linked immunosorbent assay reader (BioTek Synergy 2), using 540/570 nm as the measurement wavelength and 630 nm as the reference wavelength.

The mouse model was performed essentially as described in ref. ³⁵, from which parts of the following text were used: “All animal procedures were approved by the University of Luxembourg Animal Experimentation Ethics Committee and by appropriate government agencies. The animal work of the present study has been conducted and reported in accordance with the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines to improve the design, analysis and reporting of research using animals, maximizing information published and minimizing unnecessary studies. Three-to-four-month-old C57BL/6N male and female mice were obtained from Charles River Laboratories (France). Mice were housed in 12 h light/dark cycle, with sterile food and water ad libitum. The mice were specific pathogen free, housed in individually ventilated cages at a maximum of 5 per cage and maintained at a temperature of 22 °C and a relative humidity of 55%. They were kept on autoclaved corn cob bedding and fed with SAFE Diets irradiated at a min of 25 kGy. The watering system consisted of reverse osmosis water with 2 ppm of chlorine. Mice were treated with a single intraperitoneal injection of LPS (4 μg LPS/g body weight) or PBS as vehicle control. Mice were deeply anaesthetized with a combination of ketamine (100 mg/mL; Nimatek Vet) and dorbene (medetomidine hydrochloride; 1 mg/mL; Dorbene Vet) 0, 12, 24 and 48 h after LPS injection. Spleens were dissected following transcardiac perfusion with ice-cold PBS, collected in ice-cold HBSS (Gibco/Life Technologies) with 1 M HEPES (Gibco/Life Technologies) and 0.5% D-(+)-glucose (Sigma-Aldrich) and then stored in liquid nitrogen.”