Quantifying GAS Dissemination in Mice

6–8 week old C57BL/6 (Charles River, Margate, UK), and age and weight-matched C57BL/6 C5^-/- and C57BL/6 C3^-/-/C5^-/- female mice were challenged intra-muscularly with 1×10⁸ GAS, and quantitative endpoints compared at 3 hours post infection. Mice were euthanized, blood taken by cardiac puncture and infected thigh muscle, spleen, liver, and draining inguinal lymph nodes dissected. All organs were plated to quantify bacterial cfu and systemic dissemination. Additional C57BL/6 mice were challenged intra-muscularly with 1×10⁸ GAS. Whole thighs were formalin-fixed, and paraffin-embedded for histopathology imaging as described previously [24 (link)], or homogenized and prepared for western blotting as described above. No animals were excluded from the study and randomization was not necessary as mice were genetically identical and age, weight and sex matched. No blinding was carried out as mice were caged separately to prevent contamination with different GAS strains.