Soya peptone

Bacterial strains were grown on tryptic soy agar for 48 h at 22 °C. A single colony of each strain was inoculated into 10 mL of tryptic soy broth [Casein peptone (17 g), dipotassium hydrogen phosphate (2.5 g), glucose (2.5 g), sodium chloride (5 g), soya peptone (3 g) per liter, Sigma, Germany] and incubated for 17 h at 22 °C with shaking (150 rpm). These starter cultures were then diluted with fresh sterile tryptic soy broth to an optical density (OD 600) of 0.10 ± 0.05. Five hundred microlitres of the diluted starter cultures were inoculated in duplicates, into 25 mL of tryptic soy broth with or without iron chelator, 2,2′-bipyridyl, MW 156.18 g/mol (Sigma, Germany). Iron depletion was attained by the addition of 100 μM of 2,2′-bipyridyl to the broth. Cultures were grown overnight at 22 °C with shaking (150 rpm) until the late log phase. Cells were harvested by centrifugation at 4000 rpm for 10 min at 4 °C, then washed three times with phosphate buffered saline containing bacterial protease inhibitor cocktail (Sigma, Germany) and stored at −80 °C.

We used the rod-shaped Gram-negative bacterium B. fungorum (ATCC BAA-463) first described in Coenye et al. (2001) (link). It was found in basaltic aquifers of Snake River Plain as well as Hawaiian volcanic deposits (Sebat et al., 2003 (link); Dunfield and King, 2005 (link)) and is known to weather rocks for nutrient acquisition (Wu et al., 2007 (link); Mailloux et al., 2009 (link)). The microorganism was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Germany. A microbial strain of B. fungorum was grown for 24 h in tryptic soy broth complete medium (17 g/L casein peptone, 2.5 g/L K₂HPO₄, 2.5 g/L glucose, 5 g/L NaCl, 3 g/L soya peptone; Sigma-Aldrich) at 30°C on a shaking table. Five milliliter were removed and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, the culture washed with 5 ml M_+P and centrifuged. This step was repeated three times. The washed cells were again cultured for 24 h in 50 ml M_+P at 30°C on a shaking table. A cell density of 10⁸ cells/ml was determined by counting with a hemocytometer. Five milliliter were removed, washed with M_-P and centrifuged. This step was repeated three times. A total of 50 μl of this culture were added to each assay in order to obtain an initial cell density of 10⁵ cells/ml.

In order to analyze S. aureus-mediated induction or inhibition of MDSC formation, we used a variety of staphylococcal strains (Table 1). Bacteria were stored as glycerol stocks at −80°C and grown overnight on TSB agar plates at 37°C (casein peptone 17 g/l, soya peptone 3 g/l, glucose 2.5 g/l, dipotassium hydrogen phosphate 2.5 g/l, sodium chloride 5 g/l, Sigma-Aldrich). Single colonies from each strain were inoculated and shaken for 16 h at 130 rpm at 37°C in RPMI 1640 medium (Biochrom) supplemented with 4 mM L-glutamine (Gibco/Life Technologies). Bacterial cells were removed by centrifugation for 30 min at 5,000x g at 4°C and the supernatants were sterile-filtered twice using 0.2 μm non-pyrogenic filters. Equivalent growth of the bacteria was verified by optical density measurements at 600 nm and by CFU counting on TSB agar plates. P. aeruginosa was grown overnight in TSB medium instead of RPMI 1640, and supernatants were prepared as described (Rieber et al., 2013 (link)). The filtered supernatants were stored in aliquots at −20°C and were used for stimulation experiments.