Eon high performance microplate spectrophotometer

The degree of methylesterification was analysed by saponification of AIR followed by enzymatic oxidation of methanol released by alcohol oxidase, as described by [40 (link), 41 (link)] with modifications. Briefly, de-starched AIR (4 mg) was saponified for 1 h at room temperature by suspending in 180 µL of water and 60 µL of 1 M NaOH with shaking. Afterwards, the solution was neutralized with HCl and centrifuged. The supernatant with released methanol (50 µL) and alcohol oxidase (0.05 U in 0.1 M sodium phosphate, pH 7.5) (Sigma) was loaded into a microplate and incubated for 20 min at room temperature with shaking. Next, 100 µL of mix containing 0.02 M 2,4-pentanedione in 2 M ammonium acetate and 0.05 M acetic acid was added to each well and incubated for 10 min at 68 °C. Samples were cooled on ice and absorbance at 412 nm was measured in a microplate reader (Eon™ High-Performance Microplate Spectrophotometer, BioTek). The degree of methylesterification was calculated as the molar ratio of methanol to galacturonic acid (expressed in %). The content of galacturonic acid was quantified previously by HPAEC, as described in the “Cell wall polysaccharide compositional analysis” section.

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).

The 96-well plates (One Riverfront Plaza Corning, Corning, NY, USA) were coated with 100 ng per well of purified nvIBDV VP2 protein expressed by Pichia pastoris (P. pastoris) (Ruipu, Tianjin, China) in the coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) at 4 °C overnight and then blocked by 5% non-fat milk at room temperature for 1 h [25 (link)]. After that, 100 μL of 1:500 diluted chicken sera was added to each well of the coated plates. The plates were incubated at 37 °C for 30 min, followed by washing with PBST three times. The goat anti-chicken IgY secondary antibody HRP (Invitrogen, Corning, NY, USA) diluted with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.05% Tween-20, pH 7.4) was added, and the plates were incubated at 37 °C for another 30 min. After washing three times with PBST, the plates were visualized by adding 100 μL 3,3′,5,5′-tetramethylbenzidine solution (Tiangen, Beijing) and incubated for 10 min at room temperature. Reactions were terminated by adding 50 μL of 2 M sulfuric acid. Absorbance at 450 nm was recorded using an Eon™ High Performance Microplate Spectrophotometer (BioTek, Shoreline, WA, USA). The S/P value was calculated by the following formula: (S/P value = (sample value − mean value of NC)/(mean value of PC − mean value of NC)). A sera sample with an S/P value > 0.2 was considered positive.