Balb cj mice

BALB/cJ mice (Charles River) were kept in accordance with legal and ethical policies on animal research. The animal study was reviewed and approved by the federal authorities of Rhineland-Palatinate, Germany and all mice were kept in accordance with federal and state policies on animal research at the University of Mainz and BioNTech SE. Germline BALB/cJ DNA was extracted from mouse tail. 4T1 WT cells were purchased from ATCC. Third and 4th passages of cells were used for tumor experiments.

Six-week-old female BALB/cJ mice were purchased from Charles River (Harlow, UK) and housed at the Functional Genomics Facility, University of Oxford. Groups of 6 mice were immunized intramuscularly with 3 µg for each vaccine component, delivering between 3 µg for monovalent and 12 µg for tetravalent vaccinations at weeks 0 and 4. Mice were bled at 1 week and culled at 5 weeks post boost. On the day of sacrifice, spleens were harvested and splenocytes were isolated individually by pressing organs through a 70 µm sterile nylon-mesh cell strainer (Fisher Scientific, Waltham, MA, USA) using a 5 mL syringe rubber plunger. Following the removal of red blood cells (RBC) with RBC Lysing Buffer Hybri-Max (Sigma-Aldrich, Pool, UK), splenocytes were washed and resuspended in R10 (RPMI 1640 supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin, and β-mercaptoethanol for ELISPOT and intracellular cytokine staining (ICS) assays. All animal procedures and care were approved by the local Clinical Medicine Ethical Review Committee, University of Oxford and conformed strictly to the United Kingdom Home Office Guidelines under the Animals (Scientific Procedures) Act 1986. Experiments were conducted under project license 30/3387 held by T.H.

BALB/cJ mice were originally purchased from Charles River Laboratory. IL-25 deficient mice were a generous gift from Dr. Andrew N.J. McKenzie32 (link). These mice had been backcrossed for 10 generations onto the Balb/cJ background prior to receiving them into our facility. All mice were bred and maintained in the Benaroya Research Institute Animal Facility under specific pathogen-free conditions.

Experimental subjects were male BALB/cJ mice (USA, JAX^TM Mice stock n° 000651) provided through Charles River. Upon arrival, at 25 days of age, BALB/cJ mice were housed in 33 × 13 × 14 cm Plexiglas boxes in groups of four. We also purchased C57BL/6J mice from Charles River (Italy) to be used in the resident-intruder test (see below). C57BL/6J mice were housed in groups of 6 in 42 × 17 × 14 cm Plexiglas boxes, in the same housing room as BALB/cJ mice. Animals had access to water, environmental enrichment in the form of shelter material and food ad libitum (Mucedola, Settimo Milanese, Italy). The animal room was maintained under an inverted 12:12 h L/D cycle (lights on at 07:00 pm). Temperature (22 ± 1°C) and humidity (42–52%) were monitored and kept constant.

Splenocytes were isolated from spleens of BALB/cJ mice (Charles River, France) resuspended in DMEM supplemented with 1% FBS and seeded in a 24-well plate. Cells were pre-incubated with the test compounds for 2 h, stimulated with 1 μg/mL LPS from E. coli 0111:B4 (Sigma Chemical Corp. Saint Louis, USA) and incubated overnight. Concentration of IL-6 was determined in cell supernatants by sandwich ELISA using capture and detection antibodies (R&D Systems, Minneapolis, USA) according to the manufacturer’s recommendations.