Ficoll hypaque density gradient

Peripheral blood mononuclear cells (PBMCs) from adult healthy controls and liver mononuclear cells (LMCs) from healthy cadaveric donors and recipients were obtained using Ficoll-Hypaque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden).

Bone marrow cells were obtained by flushing the marrow cavities of the femurs and tibias with phosphate buffered saline (PBS) (pH 7.4), containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, Steinheim, Germany). Bone Marrow-derived Mononuclear cells (BMMNCs) were isolated from the buffy coat by Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden). BMMNCs were differentiated into macrophages by incubating in RPMI 1640 (Corning, Manassas, VA, USA) with supplemented 10% fetal bovine serum (PAA, Pasching, Austria), 50 ng/mL M-CSF (#315-02, Peprotech, Rocky Hill, NJ, USA) at 37 °C in 5% CO₂ for 7 days. After 7 days, Bone Marrow-derived Macrophages (BMDMs) were harvest by using 0.025% Trypsin–EDTA (Hyclone, Logan, UT, USA) and or continuing cultured for 21 days at 37 °C in 5% CO₂ in 90% RPMI 1640 (Corning, Manassas, VA, USA) with supplemented 10% fetal calf serum (PAA, Pasching, Austria) and changed every 2–3 day until 21 days.

Cell separation was done within 4 hours after collection. Mononuclear cells (MNCs) were obtained by centrifugation over Ficoll-Hypaque density gradient (1.077 g/cm³, GE Healthcare) and CD34⁺ cells were purified by using an immunomagnetic cell sorting (MACS) technology according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch-Gladbach, Germany). As well as, purity of isolated UCB-derived CD34⁺ assessed by flow cytometry.

PBMCs were isolated using the Ficoll–Hypaque density gradient method (GE Healthcare, Chicago, IL, USA) and stored in liquid nitrogen before shipping to the South African Tuberculosis Vaccine Initiative (SATVI) for processing. Cryopreserved cells were thawed and rested for two hours in Roswell Park Memorial Institute RPMI 1640 media containing 10% heat-inactivated fecal bovine serum (FBS) prior to antigen stimulation. We stimulated cells with a pool of early secretory antigen (ESAT-6) and culture filtrate protein (CFP-10, peptide pool referred to as ESCF) consisting of 17 and 16 peptides, respectively, overlapping by 10 amino acid sequences (1 µg/mL, GenScript, Piscataway, NJ, USA); Mtb cell lysate (MtbLy) (H₃₇Rv; 10 µg/mL, BEI Resources, Manassas, VA, USA), and HIV-1C Gag 15 mers overlapping by 10 amino acids (2 µg/mL, BEI Resources, Manassas, VA, USA). Phytohemagglutinin antigen (PHA; 2 µg/mL) was used as a positive control. A negative control without stimulation (NS) was also included. Stimulations were performed for two hours in a 37 °C CO₂ incubator, after which Brefeldin A (5 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added and cells were further stimulated for four hours. All stimulations were conducted in the presence of the co-stimulatory antibodies, anti-CD28 and anti-CD49d (1 µg/mL, BD Biosciences, San Jose, CA, USA) for a total of six hours.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (GE, USA). CD14+ monocytes were then isolated from the PBMCs by positive selection using magnetic beads (Miltenyi Biotec, Germany) according to the protocol provided by the manufacturer.

Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare) from the peripheral blood of healthy blood-donors, obtained from the Blood Collection Service of the Pizzardi Hospital of Bologna, according to the requirements of the local ethical committee. Macrophages were obtained by differentiation of monocytes by culture in RPMI 1640 (Sigma) medium supplemented with 20% FCS, 2 mM L-glutamine and 100 μg/mL penicillin/streptomycin. After 7 days, monocyte-derived macrophages were detached with a cell scraper and dispensed in 24 well plates at a cell density corresponding approximately to 50% of confluence. One day later, macrophages were incubated with standard unconditioned culture medium or with the media conditioned by MCR_Nc or MCR_sTn cells either BCG-challenged (as described above) or mock challenged. After 2 h, the conditioned media were replaced by fresh medium, which was collected 24 h later and stored at -80 °C for the detection of cytokines secreted by macrophages.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Uppsala, Sweden) at 400 ×g at 21°C for 30 min. The cells were then resuspended in RPMI 1640 medium (GE Healthcare) containing 50 mM HEPES buffer (Gibco, Grand Island, NY, USA), 10% inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 50 mM β-mercaptoethanol (Gibco), and 40 μg/mL gentamicin (Neoquímica, Anápolis, GO, Brazil) to a final concentration of 2 × 10⁶ cells/mL. PBMCs were cultured in 24-well microplates (Falcon, San Jose, CA, USA) in the presence of 4 μg/mL M. bovis antigen or maintained in culture medium at 37°C in a 5% CO₂ atmosphere. The cells were collected after 48 h for immunophenotyping.