PBMC-Yeast Interaction Protocol

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.