Limulus amebocyte lysate

Human PBMCs were isolated using Histopaque-1077 (Sigma) as reported (Endres et al., 1988 (link)). To stimulate cytokine production, 100 μL containing 5 × 10⁵ PBMCs in RPMI 1640 Dutch modification (added with 2 mM glutamine, 0.1 mM pyruvate and 0.05 mg/mL gentamycin; all reagents from Sigma) were plated onto round-bottom 96-well microplates, and 100 μL with 1 × 10⁵ fungal cells freshly harvested or treated were added to each well. The co-cultures were incubated for 24 h at 37°C with 5% (v/v) CO₂. In some experiments, PBMCs were pre-incubated for 1 h at 37°C with 200 μg/mL laminarin before stimulation with yeast cells. Laminarin used for the pre-incubation experiments was not contaminated with LPS (tested with the Limulus amebocyte lysate from Sigma); however, all reactions were performed in presence of 5 μg/mL polymyxin B (Sigma) (Schwartz et al., 1972 (link)). The plates were centrifuged for 10 min at 3000 × g at 4°C, and the supernatants were collected and stored at −20°C until used.

Human PBMCs were isolated by density centrifugation using Histopaque-1077 (Sigma) as described (Endres et al., 1988 (link)). For stimulation experiments, the interactions were performed in round-bottom 96-well microplates with 100 μL of cells adjusted to 5 × 10⁵ PBMCs 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 with 1 × 10⁵ fungal cells freshly harvested or treated. The interactions were incubated for 24 h at 37°C with 5% (v/v) CO₂. In some experiments, PBMCs were preincubated for 1 h at 37°C with either laminarin (200 μg/mL), anti-TLR2 (10 μg/mL, eBioscience, Cat. No. 16-9922) or antibodies to TLR4 (10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-293072) prior to stimulation with Candida cells. Isotype matched, irrelevant antibodies, IgG₂aκ (10 μg/mL, eBioscience, Cat. No. 14-4724-85) and IgG₁(10 μg/mL, Santa Cruz Biotechnology, Cat. No. sc-52003) were used as controls for experiments assessing TLR2 and TLR4, respectively. All reagents used for the pre-incubation experiments were negative to contamination with LPS (tested with the Limulus amebocyte lysate from Sigma), and all reactions were performed in presence of 5 μ g/mL polymyxin B (Sigma) (Schwartz et al., 1972 (link)). Plates were centrifuged for 10 min at 3000 × g at 4°C, the supernatant saved and kept at −20°C until used.