Bicinchoninic acid assay

All experiments to evaluate the HDL function used human umbilical vein endothelial cells (EA.hy926 cell line) (HEC) from ATCC (Manassas, VA, USA), grown after the manufacturer instructions. At confluence, after 6 h starvation (exposure to the serum free medium), HEC were pre-incubated with 80 µg protein/ml HDL₂ or HDL₃ without fetal calf serum, for 18 h. Cells were further exposed to additional 10 µM tumor necrosis factor (TNFα). In parallel, cells without HDL pre-incubation were exposed to TNFα in the same condition. After 6 h, the culture media were collected and cells were harvested by lysing them with radio-immunoprecipitation assay (RIPA) buffer. The protein level in cell lysates was quantified by bicinchoninic acid assay following the manufacturer instructions (Sigma-Aldrich Co., MO, USA).

Live adult worms were obtained from the bile ducts of bovine livers and then washed for 1 h at 37 °C with PBS (pH 7.4) and used to prepare FhESP and FhSom as previously described39 (link). Endotoxins were removed using Detoxi-Gel Endotoxin Removing Gel (Thermo Fisher Scientific Inc., Illinois). The protein concentration of parasitic lysates was measured using a bicinchoninic acid assay (Sigma-Aldrich, Missiouri). Endotoxin levels were determined using the Pyrochrome Limulus Amebocyte Lysate kit (Cape Cod Inc., Massachusetts).

PLGA 50:50 lactide:glycolide ratio (52 kDa, DL‐lactide, Lakeshore Biomaterials) was functionalized with lactobionic acid (LB, Sigma Aldrich) and fabricated from 5.5% PLGA in dichloromethane (DCM, Fisher) by a double emulsion method.²⁵ The polymer solution, containing 0.1% w/v FGF7 and 0.9% human serum albumin (HSA, Sigma Aldrich) was homogenized stirred, filtered, and freeze dried. Particle size distribution (Coulter LS230, Beckman) was measured. Protein release (HSA+FGF7) was measured by bicinchoninic acid assay (Sigma Aldrich) for 21 days.

Total protein was extracted from six developing grains at harvesting time points 13, 25 and 49 DAA using phenol extraction [99 (link)] with an extraction buffer containing: 0.7 M sucrose, 5% (w/v) SDS, 0.1 M Tris–HCl (pH 8.0), 50 mM EDTA, 20 mM DTT and 1 mM PMSF. Before extraction, the grains were ground in liquid nitrogen with 20 mg PVPP. The protein pellet resulting from the phenol extraction was solubilized in 8 M urea, 4% (w/v) CHAPS, 20 mM DTT. Total protein concentration was quantified using a Bicinchoninic acid assay (Sigma Aldrich). An aliquot from each sample, corresponding to 200 μg of protein at 25 DAA was mixed with XT sample buffer (Bio-Rad), incubated at room temperature for 1 h and electrophoresed on a precast Criterion XT 12% gel (Bio-Rad) in MOPS buffer for 1 h at 200 V. Western blots were obtained as previously described [100 (link)] using antibodies towards CYP79A1 and CYP71E1 in 1:2000 dilution.

Total cell lysates from ileal samples were prepared in ice-cold lysis buffer (500 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS, 1 mM protease inhibitor). Protein concentrations were determined by bicinchoninic acid assay (Sigma-Aldrich, Overijse, Belgium). Cell extracts were subjected to cytokine analyses using the MSD mouse pro-inflammatory V-plex assay containing antibodies for IL1β, IL10, IFNγ and TNFα. These assays were performed according to manufacturer’s instructions using the MSD Quickplex SQ 120 instrument and MSD Discovery Workbench data analysis software v4.0 (Meso Scale Discovery^TM; MSD, Rockville, MD, USA).