Ficoll histopaque 1077

Human CD14⁺ monocytes were isolated from total peripheral blood mononuclear cells of healthy donors, by immunomagnetic cell sorting. Samples from healthy donor subjects were obtained within a clinical protocol (n°463-2021), approved by the institutional Ethical Committee at IRCCS MultiMedica Milano, Milan, Italy, in accordance with the Helsinki Declaration of 1975 as revised in 2013. 30 ml of whole blood (<1 h old), collected into EDTA tubes, were subjected to Ficoll Histopaque^®-1077 (Sigma-Aldrich, Inc., Saint Louis, MO, United States) density gradient stratification at 800 × g, for 25 min, room temperature (RT). The white ring interface, enriched in mononuclear cells, was collected and washed in PBS at 300 × g for 5 min, RT. Residual red blood cells were removed by treating the cell pellets with 1X Red blood lysis buffer solution (10X stock solution: NH₄Cl 83g, KHCO₃ 10 g, EDTA 1 mM, pH 7.2–7.4 in 1 L of H₂O), incubated for 10 min at 4°C and then washed in PBS at 300 × g for 5 min, RT. Monocytes were purified from PBMCs by immunomagnetic cell sorting (positive selection) using CD14 MicroBeads UltraPure (Miltenyi Biotec, Auburn, CA, United States) according to the manufacturer’s instructions.

Twelve milliliters of heparinized whole blood, collected from healthy donors, was diluted with PBS 1:1 (v/v), then subjected to a density gradient stratification with Ficoll Histopaque-1077 (Sigma Aldrich, St. Louis, MO, USA) at 500× g for 20 min. The white ring interface, composed of total mononuclear cells (MNCs), was collected, washed twice in PBS, then used for subsequent experiments for NK cell polarization and treatments. Human samples were collected from healthy donors, enrolled within studies approved by the institutional review board ethics committees (protocol no. 0024138 04/07/2011, University of Insubria, Italy and protocol no. 10 2 10/2011, IRCCS MultiMedica, Milan, Italy) and according to the Helsinki Declaration of 1975 as revised in 2013. All subjects included in the study signed the informed consent, in accordance with the Helsinki Declaration of 1975 as revised in 2013.

The cell lines used were human peripheral blood mononuclear cells (PBMC); Sk-Mel-19, Sk-Mel-28, Sk-Mel-103 human melanoma cells; B16F10-Nex2 murine melanoma cells; and K562 and Jurkat leukemic cells. For PBMC, B16F10-Nex2, K562, and Jurkat cells, RPMI medium was used. SK-Mel cell lines were cultured in DMEM high glucose medium. All (except PBMC) were kept in bottles containing their respective culture media and supplemented with 10% fetal bovine serum for the other strains, added 1% antibiotic (penicillin/streptomycin) at 10 mg/mL, conditioned in an incubator at 37 °C and 5% CO₂.
For the preparation of human peripheral blood mononuclear cells (PBMC), human peripheral blood from a donor was collected in tubes containing sodium citrate anticoagulant. PBMC were isolated with Ficoll Histopaque-1077 (1.077 g/cm³) (Sigma Aldrich) according to the manufacturer’s instructions. The use of human blood was approved by the Ethics Committee of the Universidade Federal da Grande Dourados (UFGD) under protocol number 123/12.

Human PBMCs from venous blood were isolated using Ficoll-Histopaque-1077 (Sigma) and gradient centrifugation as described [33 (link)]. The PBMC-yeast interactions were carried out in round-bottom 96-well plates containing aliquots of 100 μL of 5 × 10⁶ PBMCs/mL in RPMI 1640 Dutch modification (added with 2 mM glutamine, 0.1 mM pyruvate, and 0.05 mg/mL gentamycin; all reagents from Sigma), and 100 μL of 1 × 10⁶ yeast cells/mL. The interactions were incubated for 24 h at 37 °C with 5% (v/v) CO₂, plates were centrifuged for 10 min at 874× g and 4 °C; the supernatants were recollected and kept at −20 °C until used [36 (link)]. The concentration of TNF-α, IL-6, and IL-10 was quantified by ELISA using the kit ABTS ELISA Development from Peprotech (Rocky Hill, NJ, USA). Mock reactions where the fungal cells were not added to the interactions were included as controls. In some experiments, PBMCs were pre-incubated for 1 h at 37 °C with 200 μg/mL laminarin (Sigma) before interaction with fungal cells.

Neutrophils were isolated from whole blood in CPTTM tubes as previously described (Heit et al., 2006 (link); Lindblom et al., 2018 (link)). Briefly, whole blood was subjected first to dextran sedimentation using 6% Dextran/0.9% NaCl solution at room temperature for 45-60 min. Supernatant containing neutrophils and mononuclear cells was transferred to a conical tube and centrifuged for 12 min at 300 RCF, 4°C with low brake. Neutrophils and mononuclear cells were separated from the erythrocyte-rich pellet by lysing erythrocytes with Ammonium-Chloride-Potassium (ACK) Lysing buffer (ThermoFisher). The pellet was resuspended in phosphate buffered saline (PBS) and the cell suspension was layered over Ficoll-Histopaque-1077 (Catalog No.1077, Sigma) and centrifuged for 30 min at 450 RCF, room temperature, low brake. After centrifugation, PBMCs were removed, and the pellet that contained neutrophils was further processed. Neutrophils were rinsed with 4 mL of Hanks’ Balance Salt Solution (HBSS), centrifuged at 450 RCF for 5 min and resuspended in 2 mL HBSS. Cell count and viability data were obtained using the MUSE cell analyzer system (Millipore). Cells were cryopreserved using 30% dimethyl sulfoxide (DMSO) in RPMI medium and thawed for later use following a previously reported protocol (Milani et al., 2016 (link)). A flowchart of the methods is shown in the Supplementary Figure S1.

Cytofluorimetric analysis was performed at Siena University respiratory diseases laboratory in the period April 2020 to April 2021. Briefly, the peripheral blood samples were collected and processed within 8 h Ficoll density gradient separation was performed through Ficoll Histopaque®-1077 (Sigma-Aldrich, Burlington, MA, USA) as previously reported Live cell counts were performed in a Burker chamber. Aliquots of 3 × 10⁶ cells were stored in liquid nitrogen in a standardised manner as previously reported [26 (link),27 (link),28 (link),29 (link),30 (link)]. All experiments were performed after thawing under the same conditions.