Siliconized tubes

Blood samples from healthy volunteers or CD-1 mice were collected in tubes containing hirudin (Roche, Switzerland) as the anticoagulant to preserve complement activity. One hundred sixty microliters of freshly drawn blood was mixed with 20 μl volumes containing S. aureus (∼1 × 10⁷ CFU/ml), S. haemolyticus (∼1 × 10⁶ CFU/ml), or E. coli (∼1 × 10⁷ CFU/ml) or 40 μl of L. lactis (∼1 × 10⁸ CFU/ml) in RPMI 1640–HSA. When applicable, 20 μl of RPMI 1640–HSA or recombinant full-length SdrD (rSdrD) (4 (link)) in RPMI 1640–HSA was added to the samples in siliconized tubes (Sigma-Aldrich, Germany) at a final concentration of 12 μg/ml. After incubation for 3 h at 37°C on a horizontal rotator, the blood cells were lysed by addition of 1 ml ice-cold H₂O supplemented with 0.3% saponin (Sigma-Aldrich, Germany). Bacterial survival was evaluated by serial dilution on blood or Todd-Hewitt broth (THB) agar plates. Percent survival was determined by comparing the number of surviving bacteria to the number of bacteria in the inoculum.

MRSA viability in human whole blood was assessed as previously described with the following modifications [19 (link)]: Freshly drawn human blood anticoagulated with heparin (160 µL) was mixed with 2 × 10⁴ CFU MRSA (20 µL) grown overnight in the absence or presence of sub-inhibitory VAN (0.5 mg/L), or grown overnight and subsequently co-incubated with VAN (0.5 mg/L), 20 μL of PBS and/or 20 μL of MVs (final concentration of 10 μg per 100 µL) in siliconized tubes (Sigma-Aldrich, Taufkirchen, Germany). Tubes were incubated for 3 h at 37 °C on a rotator. Next, serial dilutions of samples were performed using sterile ice-cold MQ water + Saponin (0.3%) (Sigma Aldrich, Taufkirchen, Germany), and were plated on Todd Hewitt Agar (THA) plates for bacterial enumeration and calculation of percentage survival vs. the initial inoculum.

GFP-E1162 and GFP-E1162ΔtirE from a blood agar plate were diluted in BHI to OD₆₀₀ 0.1 and grown at 37°C to OD₆₀₀ 0.4, washed with PBS, and resuspended in RPMI-HSA. Blood was obtained from a healthy volunteer in a hirudin blood tube (Roche Diagnostics, Switzerland) and diluted in RPMI-HSA. For all whole-blood experiments, blood was used at 80% if not otherwise indicated.
Phagocytosis was performed in round-bottom polystyrene tubes under rigorous shaking at 37°C for 20 min. Erythrocytes were lysed by adding FACS lysing solution (BD) for 20 min at 25°C. The remaining immune cells were washed with RPMI-HSA and fixed in 150 µl 1% paraformaldehyde in RPMI-HSA. Fluorescence was measured through flow cytometry (FACSCalibur; Becton Dickinson, USA).
For blood survival assays, E1162 and E1162ΔtirE were grown in BHI to OD₆₀₀ 0.4, washed with PBS, and incubated with 80% fresh hirudin blood in siliconized tubes (Sigma-Aldrich, USA) on a turning wheel at 37°C for 3 h, if not otherwise indicated. Blood cells were lysed in 0.3% saponin, and CFUs were counted on blood agar plates.