Ampicillin

To remove the commensal gut microbiota, ICR mice were transferred to sterile cages and treated by adding ampicillin (1 g/L; Ratiopharm), neomycin sulfate (1 g/L; Sigma), vancomycin (500 mg/L; Cell Pharm), and Mtz (1 g/L; Fresenius) to the drinking water casually for 2 weeks.

IL-10^−/− mice (in C57BL/10 background, B10) were bred and maintained in the facilities of the “Forschungsinstitut für Experimentelle Medizin” (FEM, Charité - Universitätsmedizin, Berlin, Germany), under specific pathogen-free (SPF) conditions.
To eradicate the commensal gut flora, mice were transferred to sterile cages and treated by adding ampicillin (1 g/L; Ratiopharm), vancomycin (500 mg/L; Cell Pharm), ciprofloxacin (200 mg/L; Bayer Vital), imipenem (250 mg/L; MSD), and metronidazole (1 g/L; Fresenius) to the drinking water ad libitum as described earlier [20] (link) starting at 3 weeks of age right after weaning. Age matched female mice were subjected to the quintuple antibiotic treatment for 3–4 months before the infection experiment.

Female C57BL/6j mice were bred and maintained under SPF conditions in the Forschungsinstitute für Experimentelle Medizin (Charité – University Medicine, Berlin, Germany). Secondary abiotic mice with a virtually depleted microbiota were generated as described previously (Heimesaat et al., 2006 (link)). In brief, 8 weeks old mice were transferred into sterile cages and subjected to a broad-spectrum antibiotic treatment for 8 weeks by adding ampicillin plus sulbactam (1 g/L; Ratiopharm, Germany), vancomycin (500 mg/L; Cell Pharm, Germany), ciprofloxacin (200 mg/L; Bayer Vital, Germany), imipenem (250 mg/L; MSD, Germany) and metronidazole (1 g/L; Fresenius, Germany) to the drinking water (ad libitum). Cultural and culture-independent (i.e., 16S rRNA based molecular) quality control measures revealed virtual absence of bacteria in fecal samples as described earlier (Ekmekciu et al., 2017 (link)).

Fresh tumor tissue was cut into pieces with a maximum diameter of 2 mm and implanted subcutaneously in anaesthetized NMRI-nu mice (RjOrl:NMRI-Foxn1^nu/Foxn1^nu, Janvier Labs SAS, Saint-Berthevin, France). After harvest from euthanized animals, tumor tissue was dissociated into a single cell suspension by the GentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) applying a standardized protocol. In brief, tissue was minced manually and subjected to repeated cycles of mechanical stirring and enzymatic digestion. The resulting suspension was filtered and centrifugalized. The pellet was gently resuspended in a medium (Airway Epithelial Cell Growth Medium with SupplementMix, PromoCell GmbH, Heidelberg, Germany) with the addition of 50 μg/ml ampicillin (Ratiopharm GmbH, Ulm, Germany). Cells were incubated at 37°C in 5% CO₂ for 4–7 days. Following that, the initial medium was gradually replaced by DMEM (Biowest, Nuaillé, France) with the addition of 10% fetal bovine serum, 14 μg/ml phosphoethanolamine, 11 μg/ml ethanolamine, 0.5 ng/ml EGF (all Biochrom AG, Berlin, Germany), 5.0 μg/ml hydrocortisone, 5.0 μg/ml insulin (Sigma-Aldrich, St. Louis, USA), 0.5 ng/ml FGF (R&D Systems Inc., Minneapolis, USA), and 50 μg/ml ampicillin.

Female mice aged 8–10 weeks were infected with 1 × 10⁴ colony forming units (CFU) of ovalbumin recombinant L. monocytogenes (LmOVA) in 100 μl sterile PBS (Thermo Fisher Scientific, Waltham, MA) via the lateral tail vein. Control mice were injected accordingly with 100 μl sterile PBS. One week after injection, both groups were treated with ampicillin (Ratiopharm, Ulm, Germany) in the drinking water (1 g/l) for 7 days and mice were given an additional week of regeneration. For the evaluation of non‐specific fetal immune activation, we re‐infected preconceptually infected female mice during pregnancy at gd 17.5 with 1 × 10⁴ CFU of LmOVA. For neonatal infection at day 7 after being born, we used 1 × 10⁴ CFU of the attenuated ovalbumin recombinant L. monocytogenes strain deleted for actA (LmOVA ΔactA) in 100 μl sterile PBS injected i.p. Control neonates were injected with 100 μl sterile PBS. Concentration of inocula used for infection experiments was verified by plating serial dilutions on TSB agar plates.

Mice were treated with an antibiotic regimen as described^{42 (link)}. Groups of mice were given a cocktail of antibiotics (ampicillin, 1 g/L, Ratiopharm; vancomycin, 500 mg/L, Cell Pharm; ciprofloxacin, 200 mg/L, Bayer Vital; imipenem, 250 mg/L, MSD; metronidazole, 1 g/L, Fresenius) added to their drinking water ad libitum for a period of eight weeks.