4.2. Murine Sepsis Infection Model

E. coli O1:K1:H7 (ATCC, Manassas, VA, USA) was prepared with a target inoculum of 2 × 10⁶ bacteria per mouse from pre-titered frozen cultures. Bacteria were washed with phosphate-buffered saline (PBS), centrifuged at 2000× g for 5 min at 4 °C and resuspended in the appropriate volume to administer 50 µL per mouse. The mice were infected on day 3 or 4 of life. The litter sizes were approximately 7–8 pups. The bacteria were inoculated subcutaneously into the subscapular region using a 28-gauge insulin needle (Covidien, Dublin, Ireland). The mice were monitored twice daily for signs of morbidity and endpoint criteria. The mice were weighed prior to the infection and 18–24 h later before euthanasia. At the terminal point for each experiment, the mice were euthanized and blood was collected in tubes containing 5 μL of 500 mM ethylenediamine tetraacetate acid (EDTA, Fisher Scientific, Fair Lawn, NJ, USA). The spleens were pooled together in 2 mL of PBS supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA, USA) for the isolation of MDSCs.