Edta monoject tubes

Peripheral blood mononuclear cell (PBMC) collection and stimulation experiments using fungal stimuli were previously described [9 (link)]. Briefly, after obtaining the volunteer's written informed consent, samples of venous blood were drawn into 10 mL EDTA Monoject tubes (Medtronic, Dublin). PBMCs were separated by density centrifugation of EDTA blood diluted 1:1 in pyrogen-free saline solution over Ficoll-Paque (Pharmacia Biotech, Uppsala). The separated cell fraction was washed twice in saline and suspended in RPMI 1640 medium supplemented with gentamicin (50 μg/mL), l-glutamine (2 mM) and pyruvate (1 mM). The cells were counted in a Coulter counter (Beckman Coulter, Pasadena), and the concentration was adjusted to 5 × 10⁶ cells/mL.

After obtaining informed consent, venous blood was drawn from the cubital vein of volunteers into 10 ml EDTA Monoject tubes (Medtronic, Dublin). The PBMCs fraction was extracted by density centrifugation of EDTA blood diluted 1:1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech, Uppsala). The PBMCs were washed twice in PBS and suspended in RPMI 1640 medium supplemented with gentamicin (10 mg/mL), l-glutamine (10 mM), and pyruvate (10 mM). The procedure was approved by the scientific ethic committee at the Radboud University Medical Center (NL42561.091.12) and the Weizmann Institutional Review board (25–1). The cells were counted and frozen until used. A day before each experiment, the cells where defrosted, washed with PBS, and suspended in medium (RPMI 1640 with l-Glutamine supplemented with 10% heat inactivated fetal bovine serum and 1 mM sodium pyruvate) and plated on untreated plates. A day after, the cells were collected from the dish. To avoid cell lost, the dish was washed with medium and the remaining cells were added to the collected cells. The cells were then manually counted with trypan blue and normalized so the same amount of cells was used for each individual (1–5×10⁵ cells per replicate, depends on the amount of live cells in the least concentrated sample).

Venous blood from the cubital vein of healthy volunteers was drawn in 10 ml EDTA Monoject tubes (Medtronic, Dublin) after obtaining written informed consent. PBMC isolation was performed as previously described (9 (link)). In short, the PBMC fraction was obtained using density centrifugation of EDTA blood diluted 1:1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech, Uppsala). Cells were then washed twice in saline and suspended in RPMI 1640 medium supplemented with gentamycin (10 mg/mL), L-glutamine (10 mM) and pyruvate (10 mM). PBMCs were counted in a Coulter counter (Beckman Coulter, Pasadena) and re-suspended in a concentration of 5 x 10⁶ cells/mL. A total of 5 x 10⁵ PBMCs were added in 100 μL to round-bottom 96-well plates (Greiner) and incubated with 100 μL of stimulus (heat-killed Candida albicans yeast of strain ATCC MYA-3573, UC 820, 1 x 10⁶/mL) or RPMI 1640 medium. After 24 hours, the supernatants were collected and stored at −20°C until assayed using the proximity extension assay (PEA) that is being used from Olink Proteomics (https://www.olink.com/).