Rmpi 1640

A375 melanoma cells (at passage 5) and normal human epidermal melanocytes (NHEM) were purchased from American Type Culture Collection (ATCC^© CTRL-1619^TM) (Rockville, MD, USA). Cell lines were cultured under a humidified atmosphere of 5%CO₂ and passage in growth medium: RMPI 1640 (Sigma-Aldrich) containing 10% FBS (fetal bovine serum) (Sigma-Aldrich; Merck Millipore) supplemented with antibiotics (1% penicillin/streptomycin) and 1% glutamine. Once 70–80% confluence was reached, cells were harvested using trypsin, washed and counted on a microscope using a Burker hemocytometer.
Primary HUVEC (at passage 10) were kindly provided from Prof. Bazzoni of the University of Verona. Cells were cultured in a 75 cm² culture flask coated with Matrigel (Corning Incorporated; NY, USA) in the presence of M200 medium and 10% of LSGS supplement (Thermo Fisher scientific, Waltham, MA, USA) under a humidified atmosphere of 5% CO₂. Co-cultures for CD105 gene and CD31 gene/protein expression analyses, as well as for net-like structure detection, were performed using 6 wells plate Transwell with 0.4 µm pores (Corning Incorporated; NY, USA). HUVEC cells were plated in the chamber, in presence of M200 supplemented medium, while in the permeable membrane were plated A375, 3G8 Crisped clone and NHEM cells using RPMI + 10% FBS. Each co-culture was performed in three replicates.

We used A375 (American Type Culture Collection; ATCC: CTRL-1619TM) and MELHO (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen) human melanoma cells. The RUNT KO cells were obtained using CRISPR/Cas9 as we previously described [21 (link)]. Cell lines were cultured under 5% CO₂ and in RMPI (1640 (Roswell Park Memorial Institute) growth medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), supplemented with antibiotics (1% penicillin/streptomycin) and 1% glutamine. All cell lines were tested negative for mycoplasma using the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich).
Once 80% confluence was reached, cells were harvested, washed and counted using a Burker haemocytometer for all experiments.

BALB/c mice were implanted with 4T1 cells and lungs were harvested 8–9 days post-last injection for metastatic nodule quantification. In brief, lungs were harvested in ice-cold 1xPBS, minced into small pieces then transferred into digestion solution consisting of 2 mg/mL collagenase type V (Worthington) supplemented with 50 ug/mL DNAse (Sigma) and incubated for 2 h in a 37C incubator with end-over-end rotation. Suspension was transferred into 70-um strainer, washed once in 1 × PBS then transferred into 10 mL selection media consisting of RMPI 1640 supplemented with 10% FBS, penicillin–streptomycin and 10 ug/mL 6-thioguanine (Sigma). Three to four 1:10 serial dilutions were plated either in 6-well plates or 10-cm dishes and cultured for 10–14 days at 37C, 5% CO₂. Metastatic plaques were then fixed in methanol for 5 min at room temperature, re-hydrated in distilled water then stained with 0.03% methylene blue (Sigma) for 5 min at room temperature. Dye was then discarded, plate was rinsed gently with distilled water and allowed to air-dry prior to counting plaques.

pLAg was prepared by suspending promastigotes of the L. panamensis strain MHOM/COL/81/L13 at a concentration of 1 × 10⁷ parasites/mL, freezing in liquid nitrogen and thawing at 37°C four times. Cells were suspended in RMPI 1640 (Sigma-Aldrich) with 10% FBS (Gibco, Carlsbad, CA), 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL) and distributed in 96 well plates in 200 μL per well. For PBMC cultures, 4 × 10⁵ PBMCs and for B cell/CD4 T cell co-cultures, 2 × 10⁵ B cells or 2 × 10⁵ T cells or both cell types were plated per well. Then 8 μL of pLAg were added to the appropriate wells to reach a parasite:cell ratio of 0.2:1. The cells were incubated for 5 days at 37°C with 5% CO₂. Cells and supernatants were harvested for evaluation of cell surface markers and cytokine secretion, respectively.
Ramos cells were cultured in RPMI 1640 with 10% heat-inactivated FBS at a concentration of 10⁶ cells/mL with pLAg (10⁷ parasites/mL final concentration), 5 μM CpG ODN 2006 (InvivoGen, San Diego, CA) or no stimulus for 48 hours. Cells were harvested for evaluation of cell surface markers or BCR-mediated endocytosis.

NIH-3T3 fibroblasts (CRL-1658) and Daoy
(HTB-186) were obtained from the ATCC (Manassas, Virginia, US) and
cultured following the provider’s guidelines using Dulbecco’s
modified Eagle medium (DMEM) (Gibco, Thermo Fisher Scientific Inc.,
Waltham, MA, USA), 10% calf serum (ATCC), and Eagle’s minimum
essential medium supplemented with 10% fetal serum. RPE (ATCC, CRL-4000,
human, female), A549, and H2009 were maintained in DMEM:F12 (Thermo
Fisher Scientific). H358 cells were cultured in RPMI 1640 (Thermo
Fisher Scientific). Jurkat clone E6-1 (TIB-152, human male) were grown
in RMPI 1640 supplemented with 10% inactivated fetal bovine serum
(Sigma), 1 mM sodium pyruvate, and 10 mM HEPES (both from Sigma).
HEK-293FT and human primary fibroblasts were maintained in DMEM. All
media were supplemented with 2 mM l-glutamine and 100 units/mL
of penicillin–0.1 mg/mL streptomycin (Sigma, Merck KGaA, Darmstadt,
Germany), and unless stated otherwise, with 10% fetal serum. All cell
lines were grown at 37 °C and 5% CO₂.

Birds were sacrificed (20 birds per treatment) as per the slaughter principles covered under KS 1174/1999^{4 (link)}, and blood was collected in heparinized tubes from wing vein using gauge needles from birds from each treatment. Plasma and serum were separated by centrifugation and stored at − 20 °C until analysis. The spleen was removed under aseptic condition and weighed. The spleens were placed in cell culture medium (CCM) on ice. The CCM comprised RMPI-1640 (Sigma-Aldrich, Gillingham, UK) supplemented with 10% fetal calf serum, two mM glutamine, and antibiotics. The spleen was cleaned from all adherent tissues.

For the experiments Dulbecco's phosphate-buffered saline (D-PBS), ethylenediaminetetraacetic acid (EDTA), lipopolysaccharide from Escherichia coli (LPS), Zymosan A from Saccharomyces cerevisiae and the components of complete Growth Medium (cGM, consisting of RMPI-1640 with glutamine, 0.1 mM non-essential amino acids, 50 μM β-mercaptoethanol, 1 mM pyruvate and penicillin/streptomycin) were from Sigma-Aldrich Ltd. (Budapest, Hungary). Ficoll-Paque was obtained from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). AmBisome was purchased from Gilead Sciences Ltd. (Paris, France). The content of the vial, after reconstituting with 12 ml sterile water for injection, contained hydrogenated soy phospholipid (HSPC), 17.75 mg/mL; distearoyl-phosphatidylglycerol (DSPG), 7 mg/ml, amphotericin B, 4.2 mg/ml; cholesterol, 4.3 mg/ml; tocopherol, 0. 05 mg/ml; Sucrose, 75 mg/ml; Sodium succinate, 2.3 mg/ml. The 96-well cell culturing plates (U plate) were obtained from Sarstedt (Nümbrecht, Germany).