Nh4ch3coo

The Zn (II), Pb (II), Cd (II) and Hg (II) solutions were obtained from certified standard solutions at 1000 mg/L (Fluka Trace Select). Three different stocks solutions of Zn, Pb and Cd were prepared from cascade dilutions in order to obtain final concentrations of 10⁻⁴ M, 10⁻⁵ M and 10⁻⁶ M.
The ionic strength was set using a solution of 1 M NaNO₃ prepared from solid powder (Sigma-Aldrich, supra-pur, Saint Louis, MO, USA). The pH was adjusted with 1 M NaOH standard (Merck titripur^®, Darmstadt, Germany) or 1 M HNO₃ diluted from a 65% (Merck suprapur) solution.
For the working electrode pretreatment, a cleaning solution with 0.2 M H₂SO₄ (Sigma-Aldrich, p.a, Saint Louis, MO, USA). A second cleaning step in a solution containing 1 M NH₄CH₃COO from the salt (Sigma Aldrich p.a) diluted in 0.5 M HCl (Merck, p.a, Darmstadt, Germany) was also carried out. To prepare the solution for the redissolution of the mercury film, 0.1 M NH₄SCN from the salt (Sigma Aldrich, p.a) was used. Ultrapure milli-Q water (resistivity 18.2 MΩ cm, Elga, labwater, High Wycombe, UK) was employed in all the experiments. Nitrogen (>99.999% pure) for the solution purging was purchased from Air liquid, France.

A 0.5 × 20 cm column was packaged with 6 ml of the BioGel P4 resin (Bio-Rad) and conditioned with a 25 mM NH₄CH₃COO (Sigma–Aldrich) buffer. Ten nanomoles of the total fOS were loaded into the column and 24 fractions (500 ul per fraction) were collected. Sample were dried in vacuum and later resuspended in water and desalted by ion exchange chromatography. Ten percent of each fraction was collected for FACE analysis, and the remaining 90% was divided in half and used for macrophage stimulation in biological duplicates. For the mannosidase digestion, 10 nmol of fOS or the synthetic ManGlcNAc disaccharide were treated with 40 U of α1-6 mannosidase (New England Biolabs) and 32 U of α1-2, 3 mannosidase (New England Biolabs). Samples were then mixed with 3 volumes of cold ethanol and centrifuged at 20,000 × g for 20 min at 4 °C. The supernatant was dried in vacuum and later resuspended in water and desalted by ion exchange chromatography. Ten percent of the digested product was collected for FACE analysis and the remaining 90% was divided in half and used for macrophage stimulation in biological duplicates.