Thalidomide

Eagle’s Minimum Essential Medium (EMEM) basal cell culture medium, RPMI-1640 basal cell culture medium and foetal bovine serum (FBS) were purchased from Life Technologies Ltd. (Paisley, U.K.). All chemical reagents used as described in subsequent sections were of the highest quality available and were sourced from Sigma-Aldrich Company Ltd. (Dorset, U.K.). Combretastatin A4, thalidomide and tranilast were sourced from Abcam plc. (Cambridge, U.K.). OGT 2115 was sourced from Tocris Bioscience (Bristol, U.K.). All anti-angiogenic drug compounds were dissolved in dimethyl sulphoxide (DMSO) to produce stock solutions of 10mM. Stock solutions were diluted in either cell culture medium or the appropriate assay buffer in order to achieve the desired final concentrations used during each experimental procedure.

Purified neutrophils (> 95% of the cells) were resuspended in RPMI medium 1640 (GIBCO Life technologies, MA, USA) and (1x10⁶) incubated at 37°C in centrifuge microtubes (Eppendorf, NY, USA) with or without M. leprae whole-cell sonicate (MLWCS; 20 μg/ml; NR-19329; Bei Resources, VA, USA), CpG-Hlp complex (rHlp: 0.25 μM; CpG 2395: 0.5 μM; Invivogen, CA, USA) or phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich). The preparation of the CpG-Hlp complex was performed as previously described [12 (link)]. In the case of in vitro treatment with thalidomide (Abcam, CB, UK), the drug was dissolved in DMSO (Sigma-Aldrich, MO, EUA) and used at a final concentration of 50 μg/ml. NETs were quantified in the culture supernatant via the Quant-iT PicoGreen dsDNA Assay kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. In vitro thalidomide efficacy was tested in human monocytes stimulated with LPS (Sigma-Aldrich), as previously described [17 (link)]. Neutrophil supernatants were also tested for lactate dehydrogenase activity (LDH) by using the Liquiform LDH kit (LABTEST, MG, Brazil) according to the manufacturer’s instructions. NADH oxidation was monitored at 340 nm in the Eon High Performance Microplate Spectrophotometer (BIOTEK, VT, USA).