Lymphoprep reagent

PBMC were obtained from Ficoll separation of whole blood using Lymphoprep reagent (Stemcell Technologies). Neutrophils were subsequently isolated from the granulocyte and red cell pellet formed at the bottom of the Ficoll tube by hypotonic cell lysis in ammonium chloride buffer. Cell viability was assessed by trypan blue dye exclusion. Purified cells were visualised and counted using a haemocytometer before re-suspension in TRIzol reagent (Invitrogen, Paisley, UK). Care was taken to minimize the time between blood-drawing and placing cells in TRIzol reagent to within 3 h.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy adult donors under local ethics approval (09/H0606/71). Samples were diluted and centrifuged in a density gradient using a Lymphoprep™ reagent (STEMCELL Technologies Inc.). The CD14⁺ cells were separated with a MACS MicroBead (Miltenyi Biotec) magnetic separation system according to the manufacturer’s protocol. Subsequently, the monocytes were cultured in a 1 × 10⁶/ml density in the RPMI medium supplemented with 10% fetal calf serum, penicillin/streptomycin mix and 2 mM L-glutamine, with addition of cytokines: 250 ng/ml GM-CSF, 100 ng/ml IL-4, 10 ng/ml TGF-β1, all obtained from Pepro-Tech. After 5 days of culture, the cells were exposed to a Ni(NO₃)₂ solution (1 mM), peptides (50 µM) or Ni²⁺-peptide complexes with the same concentrations of peptides and of the nickel salt. After 48 h the cells were harvested for the flow cytometry analysis.

Buffy coats from healthy blood donors were received from the blood center at the Uppsala University Hospital, Sweden. Peripheral blood mononuclear cells (PBMC) were isolated using SepMate tubes-50 (Stem Cell Technologies) by density gradient centrifugation. Briefly, 10 ml Lymphoprep reagent (Stem Cell Technologies) was added to the tubes followed by addition of blood on top of Lymphoprep. The tubes were then centrifuged at 1200×g for 10 min. Next, cell suspension above the Lymphoprep was collected and PBMCs were washed twice with phosphate-buffered saline (PBS, Thermo Fisher Scientific). For optimal lysis of red blood cells, 5 ml ACK lysis buffer (Thermo Fisher Scientific) was added to the cells and incubated in the dark for 10 min at room temperature followed by centrifugation at 500 × g for 5 min. After that, primary monocytes were removed by an EasySep CD14+ selection kit II (Stem Cell Technology) according to the manufacturer’s instructions. Primary human lymphocytes were stored in −150 °C until use.

Peripheral blood mononuclear cells (PBMC) and plasma were isolated from up to 20 ml of venous blood by density gradient centrifugation, using standardized protocols and Lymphoprep reagent from Stemcell (Cambridge, MA, United States). Plasma was then stored at −80°C until use (10 (link)). A minimum of 1.5 million CD14+ monocytes were sorted starting from 20 million PBMC through immunomagnetic separation technique (Miltenyi Biotec, Bergish Gladbach, Germany). Purity of monocyte population was always >95%.

Leukocyte cones were obtained from anonymous healthy blood donors, in accordance with the Human Tissue Act (NHS Blood Transfusion Unit, approval number M153). In order to isolate primary human peripheral blood mononuclear cells (PBMCs), blood products were first diluted in 50 ml PBS and laid carefully on top of 10 ml Lymphoprep reagent (StemCell Technology, Vancouver, Canada). The tubes were then centrifuged at 800 g for 20 min with the brakes off. Next, PBMCs were harvested from the interface above the Lymphoprep and washed twice in 50 ml PBS. In some cases, the cells were incubated in 5 ml ACK lysis buffer (Thermo Fisher Scientific, Massachusetts, USA) at room temperature for 3 min to remove the remaining red blood cells. Primary human monocytes were then depleted using EasySep CD14+ selection kit II (StemCell Technology, Vancouver, Canada) following the manufacturer’s instructions. The resulted primary lymphocytes were then resuspended in 3 ml PBS containing 1.5 μM CellTrace CFSE or CellTrace Violet Cell Proliferation Dye (Thermo Fisher Scientific, Massachusetts, USA), incubated at room temperature for 5 min and washed twice with 10 ml PBS. The ‘assay-ready’ lymphocytes were frozen at up to 100 million cells per vial and stored in liquid nitrogen tanks.