Hirudin monovettes

Venous blood of healthy volunteers was collected in hirudin Monovettes (Sarstedt). Overnight C. glabrata YPD cultures were harvested and washed twice with sterile water. Yeasts (1 × 10⁶ in 10 µl) were added to 990 µl freshly drawn human blood and incubated using gentle rolling at 37°C. The initial inoculum (0 h) was determined, and after 30, 60, 120, and 240 min, 10 µl of infected blood sample was diluted and plated onto YPD agar (or SD supplemented with 500 µM in FeCl₃ for the aft1Δ mutant) for CFU determination in technical triplicate. This assay was performed independently with three healthy blood donors.

For mouse whole-blood models, animals were euthanized by CO₂ asphyxiation and cardiac puncture was performed with 20 µl per ml of blood of lepirudin (Refludan, Bayer), which does not interfere with complement (24 (link)), as an anticoagulant. Human whole blood was obtained from healthy adult volunteers without signs of chronic or acute disease or inflammatory conditions by venipuncture using hirudin Monovettes (Sarstedt) to avoid clotting without impairing complement integrity. Blood samples were used immediately for experiments. Where indicated, C5aR1 antagonists were added immediately to the blood followed by incubation for 10 min prior to infection. For infections, bacterial inocula were adjusted in PBS, with 1 part added to 10 parts of blood. Samples were incubated rotating over top at 37°C.