Streptomycin

All experimental procedures were performed following institutional and international guidelines and were approved by the local animal welfare authorities (LAGeSo, Berlin, Germany, T-CH0019/20). Timed-pregnant Wistar rat dams were obtained through Janvier Labs (Le Genest-Saint-Isle, France) and housed in individual cages under controlled environmental conditions with ad libitum access to food and water. At embryonic day 18 (E18), adult rats were anesthetized with isoflurane and sacrificed. Pups were segregated from the uterus and transferred into ice-cold phosphate-buffered saline. Hearts were removed and cells were isolated using the Pierce Cardiomyocyte Isolation Kit (Thermo Scientific, Rockford, IL, USA, cat. 88281) following the manufacturer’s instructions. Cells were plated on 35 mm Petri dishes in a density of approximately 2.5x10⁵ cells/cm² and grown in DMEM (Thermo Scientific) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Bio&SELL) at 37 °C in a 5% CO₂ humidified atmosphere for 7 days. The medium was changed every 2–3 days.

The following chemicals were used: Dulbecco’s MEM, sodium chloride, 2-Mercaptoethanol (Merck KGaA, Darmstadt, Germany), FBS superior, L-glutamine, Penicillin (10,000 U/mL)/streptomycin (10,000 U/mL), Trypsin/EDTA solution (Bio & Sell GmbH, Feucht, Germany), Dimethyl sulfoxide (DMSO), HEPES, Potassium chloride, sodium hydroxide, calcium chloride dehydrate, D-glucose (VWR International GmbH, Darmstadt, Germany), D-luciferin (beetle) monosodium salt (Promega, Madison, WI, USA), Bordetella pertussis toxin, L-Glutamic acid monosodium salt monohydrate (Santa Cruz Biotechnology, Dallas, TX, USA), Forskolin (Biomol GmbH, Hamburg, Germany), Lactisole (Cayman Chemicals, Ann Arbor, MI, USA), fMLF (Tokio Chemical Industry, Tokyo, Japan), mGluR2 antagonist 1 (CAS: 1432728-49-8; Biozol GmbH, Eching, Germany), metabotropic glutamate receptor 2/3 agonist LY379268 (Tocris Bioscience, Bristiol, UK), Gibco RPMI 1640 containing L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA), Probenecid (Sigma-Aldrich, St. Louis, MO, USA), Pluronic^® F-127 (AAT Bioquest Inc., Sunnyvale, CA, USA), Fluo-4 AM (Bio-Techne, Minneapolis, MN, USA). Calcium buffer was composed of 140 mM NaCl, 20 mM HEPES, 5 mM KCl, 1.8 mM CaCl₂, and 0.5 mM D-glucose, adjusted pH 7.4.

Leukocyte concentrates obtained from healthy adult blood donors were kindly provided by the Dept. of Transfusion Medicine, University Hospital Schleswig-Holstein (UKSH) Campus Kiel. Informed consent was provided by all blood donors, and the use of leukocytes for research was approved by the Ethics Committee of the Medical Faculty (code D405/10). Monocytes were isolated from Ficoll-Hypaque separated PBMC by negative magnetic selection using the Pan Monocyte (order no. 130-096-537) or the Classical Monocyte (order no. 130-117-337) kits from Miltenyi Biotec (Bergisch Gladbach, Germany), or the EasySep Human Monocyte kit from StemCell Technologies (Cologne, Germany) according to the manufacturer’s protocols. Monocytes were differentiated for 6 days in the presence of 50 ng/mL M-CSF (ImmunoTools, Friesoythe, Germany). Fresh M-CSF was added after 4 days. Freshly isolated monocytes and M-CSF-differentiated macrophages were cultured at 10⁵ cells per well in round-bottom 96-well microtiter plates in RPMI 1640 medium (GIBCO) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% heat-inactivated low endotoxin fetal bovine serum (Bio&Sell, Feucht, Germany). In some experiments, monensin (3 μM) was added to monocytes cultures for 4 h before processing for intracellular IL-10 staining.