Ficoll paque gradient

Human AML cell line KG-1 (obtained from the ATCC) was maintained in culture, splitting every 2–3 days, in Advanced RPMI medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Biosera), 2 mM L-glutamine, 50 IU/ml of penicillin and 50 μg/ml of streptomycin (Lonza).
For primary acute myeloid leukemia samples, PB or BM samples from adult or pediatric patients were collected at diagnosis. Samples were enriched for mononuclear cells by using a Ficoll-Paque gradient, and subsequently frozen in 10% dimethyl sulfoxide solution (Sigma-Aldrich). Details of patients’ samples are provided in Fig. 3.

MSCs were generated from bone marrow aspirates of rabbits (12 weeks old, 2.5–3.0 kg). According to previous methods [20 ], mononuclear cells were separated by centrifugation in a Ficoll-Paque gradient (Sigma Co., St. Louis) and suspended in 20 mL of Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS) (HyClone Logan, Utah). Cultures were incubated at 37°C and 5% carbon dioxide for 72 hours; then the nonadherent cells were removed by changing medium. When reaching 70–80% confluence, adherent cells were detached from the flask using 0.25% trypsin and subcultured. A homogenous MSCs' population was obtained after 2 weeks of culture and cells of passage 3 were harvested for further use. The adipogenic, osteogenic, and chondrogenic differentiations of cells were tested to ensure the multilineage potential.

Samples of 8 mL of maternal blood were collected in the morning at 36th week of pregnancy, prior to the beginning of labor. The umbilical cord blood was intradelivery-collected shortly after clamping. The maternal blood and umbilical cord blood were collected in Vacutainer tubes (Becton Dickinson, USA) treated with anticoagulant (EDTA). We centrifuged them at 160 G for 15 min to separate plasma from the cells. The plasma was stored at −80°C for later cytokines analysis. The isolation procedure was performed by centrifugation (40 min at 160 ×g), with Ficoll-Paque gradient (density 1.077 g/L; Sigma Chemical, St. Louis, USA). After centrifugation the supernatant was discarded, and the opaque bands at the interfaces between plasma and Ficoll-Paque-1077 containing cells were collected carefully by aspiration with a siliconized Pasteur pipet and transferred to tubes. NK cells were purified by positive selection with magnetic beads [21 (link)]. Cells were resuspended independently in serum-free medium 199 and used immediately for assays of flow cytometer.

Buffy coats were collected from healthy volunteers from Uniklinik RWTH Aachen, Germany, according to local regulations and peripheral blood mononuclear cells (PBMCs) were isolated using ficoll-paque gradient (Sigma). Subsequently, CD14⁺ monocytes were positively selected using CD14 MicroBeads (Miltenyi) according to the manufacturer’s protocol. Monocytes were pooled from 6-8 donors and plated in 96 well plates (BD#353219, black optical imaging plates) at a density of 75,000 cells/well in RPMI1640 (Thermofisher) supplemented with 10% FCS and 1% PenStrep (Gibco) and cultured in a controlled environment (37°C, 5% CO₂). Monocytes were differentiated to macrophages using 100 ng/ml recombinant human macrophage colony-stimulating factor (rh-MCSF, Immunotools) or 5 ng/ml recombinant human granulocyte-macrophages colony-stimulating factor (rh-GMCSF, Immunotools) for 7 days.

Peripheral blood samples were collected in sodium heparin tubes, and after centrifugation at 1,000 x g, 10°C for 10 minutes, plasma samples were separated and stored at -80°C. Cells were reconstituted 1:1 (v/v) in RPMI 1640 (Sigma-Aldrich, St Louis, MO, USA) and the peripheral blood mononuclear cells (PBMC) were isolated by slowly transferring blood diluted in phosphate-buffered saline (PBS) (1:2) on top of Ficoll–Paque gradient (Sigma). Samples were centrifugated at 700 x g for 40 min without break. Subsequently, the PBMC layers were collected, washed, and cell concentrations and viability were determined using the Countess II FL automated cell counter (Invitrogen Thermo Fisher Scientific). PBMC were frozen in fetal bovine serum (FBS, Gibco) with 10% dimethyl sulfoxide (DMSO; SIGMA) at -80°C for 24 hours using a freezing container (Mr. Frosty™, Thermo Scientific) and then maintained in liquid nitrogen until use.

Peripheral blood mononuclear cells were isolated from the fresh blood samples of SLE patients and HC using Ficoll-Paque gradient (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The molecular phenotypes of NK cells were detected immediately using flow cytometry analysis. The following monoclonal antibodies were used: ECD-conjugated anti-CD3, PC7-conjugated anti-CD56 (cat. no. A07748, cat. no. A21692, Beckman Coulter, Inc., Brea, California, USA), and PE-conjugated anti-TIM-3, FITC-conjugated anti-PD-1 (cat. no. 85-12-3109-42, cat. no. 85-11-9969-42, MIH clones; eBioscience, Thermo Fisher Scientific, Inc., San Diego, California, USA). The NK cells were identified as CD56⁺CD3⁻ populations. Cells incubated with PE-conjugated mouse Immunoglobulin G (IgG) or FITC-conjugated mouse IgG antibodies (cat. no. A07796, cat. no. A07795, Beckman Coulter, Inc., Brea, California, USA) were used as isotype controls. All the cell suspensions with antibodies were incubated for 30 min on ice. Data were acquired on a CYTOMICS FC 500 flow cytometer (Beckman Coulter, Inc.) and analyzed using the associated software program (CXP 2.0, Beckman Coulter, Inc.).