175 cm2 culture flasks

R. typhi (strain Wilmington, accession no. AE017197) was cultured in L929 mouse fibroblasts (ATCC CCL-1) in RPMI1640 (PAA, Cölbe, Germany) supplemented with 10% FCS (PAA, Cölbe, Germany), 2 mM L-glutamine (PAA, Cölbe, Germany) and 10 mM HEPES (PAA, Cölbe, Germany) without antibiotics (standard culture medium). 1×10⁷ γ-irradiated (1966 rad) L929 cells were seeded in 175 cm² culture flasks (Greiner Bio-One, Frickenhausen, Germany). One day later cells were infected with R. typhi and incubated for 5 to 7 days. For the preparation of bacterial stocks, infected L929 cells were resuspended in 1.5 ml PBS. 200 μl silicium particles (60/90 grit silicon carbide; Lortone inc., Mukilteo, USA) were added and cells were vortexed thoroughly for 1 min. The crude lysate was strained through a 2 μm cell strainer (Puradisc 25 syringe filter 2 μm; GE Healthcare Life Sciences, Freiburg, Germany). Bacteria were centrifuged at 4300×g for 5 min at room temperature and frozen in FCS with 7.5% DMSO in liquid nitrogen in Cryo.S tubes (Greiner Bio-One, Frickenhausen, Germany). Thawed bacterial stocks were centrifuged at 6200×g for 5 min at room temperature, washed twice with PBS and analyzed for bacterial content by quantitative real-time PCR (qPCR) and immunofocus assay as described previously [48 (link)] to determine spot forming units (sfu).

Cells grown in 175 cm² culture flasks (Greiner) were harvested 30 min after treatment by scraping, collected by centrifugation (5 min, 1200 rpm), and frozen in liquid nitrogen. The samples were thawed, mixed with double distilled 69% nitric acid and 1 mL of 30% hydrogen peroxide of Suprapur^® grade (Merck), and digested by closed-vessel microwave pressure digestion (ETHOS LEAN, Milestone), using the preprogrammed digestion program for animal products (35 min, 160–200 °C). After digestion, the samples were dried at 120 °C for two days and diluted in double-distilled 5% nitric acid. Copper concentrations were determined by ICP-OES using a Varian 720-ES (Varian Inc., Palo Alto, CA, USA).

ASCs were isolated from human tissues. Here 8 tissue samples were obtained from 7 female and 1 male donor ranging from 24 to 67 years old (46.12±15.25 yrs; average ± SD). Samples were taken from different body areas including abdomen, flanks and legs. Smokers were discarded from this study.
Samples were digested using sterile filtered 0.1% collagenase A solution (Roche, Grenzach-Wyhlen, Germany) in phosphate buffered saline (PBS, Biochrom, Berlin, Germany) incubated at 37°C until a fat emulsion emerged while inverting every 10 minutes. Culture medium (Dulbecco's Modified Eagle Medium (DMEM), 10% fetal calf serum (FCS), 10 U/ml penicillin, 10 mg/ml streptomycin sulfate, and 25 μg/ml amphotericin B) was added to stop collagenase digestion, mixed gently, and centrifuged (1300 rpm, 10 minutes, 20°C). After centrifugation the supernatant was aspirated and the pellet was suspended in 5 ml PBS and filtered through a 100 μm pore size cell strainer. Then, the sample was centrifuged again as before and the supernatant was discarded. The pellet was suspended in culture medium, transferred to 175 cm² culture flasks (Greiner Bio-One, Frickenhausen, Germany) and cultivated at 37°C and 5% CO₂. The medium was replaced the following day to remove non-adherent cells. Afterwards the medium was replaced once a week. Cells were passaged after reaching 80-90% confluence.