Nutridoma sp

hMSCs isolated from bone marrow of healthy persons under informed consent were purchased from Lonza (Cologne, Germany). Each hMSC lot used in the study was tested by Lonza for purity and their ability to differentiate into the osteogenic, chondrogenic, and adipogenic lineage. The cells were positive for CD29, CD44, CD105, and CD166, and negative for CD14, CD34, and CD45. hMSCs were cultured as described previously,^{28 (link)} using the StemMACS expansion media with a weekly medium change (Miltenyi Biotec, Bergisch Gladbach, Germany). All studies were carried out with hMSCs between the fifth and seventh passage of cultivation which exhibited an average cell doubling time of 96 h. For experiments under serum-free conditions, hMSCs were washed with serum-free medium and incubated in Dulbecco Modified Eagle Medium (DMEM) (PAA Laboratories, Coelbe, Germany) supplemented with 1% Nutridoma SP (Roche Applied Science, Mannheim, Germany) in the absence or presence of LL-37 (Anaspec, Fremont, CA, USA) at physiological concentrations of 10–100 ng/mL LL-37 as determined in human blood plasma.^{30 (link)} Cells were routinely tested for mycoplasma contamination. Cell viability was determined by trypan blue staining and periodically by application of the WST-8 assay (Dojindo, Rockville, MD, USA).

BV2 WT and mutant cells were recovered by trypsinization and resuspended in DMEM-Ham F-12 (1:1) supplemented with 1% Nutridoma–SP (Roche 11011375,001; lipid free synthetic serum complement) at a concentration of 10⁶ cells/mL. Filipin III (Merck F4767) binding kinetics (256 s) were performed on a LSR II UV flow cytometer (Becton Dickinson) equipped with a 355 nm UV laser line. Emitted fluorescence was filtered by a 450 nm+/−25 nm dichroic mirror. Filipin III (final concentration of 1 μg/mL) was added to the cells after a 60 s-recording of the natural fluorescence baseline. Cells were maintained in a 37°C water bath throughout the experiment.

Schneider’s insect medium, N-(1-naphthyl)-ethylenediamine and p-Aminobenzene-sulfanilamide were purchased from SIGMA (St. Louis, MO). RPMI 1640 medium and L-glutamine, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA, USA). Nutridoma-SP was from Roche (Indianapolis, In, USA). A23187 calcium ionophore was from Calbiochem Novabiochem Corp. (La Jolla, CA). NS-398, PGE₂ and LTB₄ enzyme-linked immunoassay (EIA) Kits were from Cayman Chemical (Ann Arbor, MI). Dimethylsulfoxide (DMSO) was purchased from ACROS Organics (New Jersey, NJ).

Siglec‐Fcs were produced as described in Padler‐Karavani et al. (2014). Siglec‐Fc vectors were transfected into HEK293A cells cultured in serum‐free media supplemented with Nutridoma‐SP (Roche). Culture supernatants were collected, and Siglec‐Fcs were purified on a Sepharose Protein A Column (GE Healthcare Life Sciences). After washing with Tris‐buffered saline (20 mM Tris–HCl, 150 mM NaCl, pH 8.0; TBS), Siglec‐Fcs were desialylated on column by neuraminidase from Arthrobacter ureafaciens (Sigma‐Aldrich) for 1 hr at room temperature. After extensive washing with TBS, Siglec‐Fcs were eluted with 0.1 M glycine–HCl pH 3.0 and pH is neutralized immediately. Siglec‐Fcs are concentrated by Amicon centrifugal filters (Millipore). The functionality of Siglec‐Fcs has been pretested on sialoglycan arrays.

Cell populations enriched for T lymphocytes from mice of the six experimental groups were obtained by nylon wool filtration of unfractionated splenic cell suspensions as previously described (Freire-de-Lima et al., 2000 (link)). T cells were cultured in DMEM supplemented with 2 mM glutamine, 5 × 10^-5 M 2-Mercaptoethanol (2-ME), 10 μg/mL gentamicin, 1 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids (all from Gibco^TM, Invitrogen Corporation) plus 1% Nutridoma-SP (Roche, Germany) instead of FBS (Nunes et al., 2013 (link)).