Macrophage and Neutrophil Depletion in Staphylococcus aureus Infection

Macrophages were depleted using clodronate liposomes (NvR). The mice were injected i.v. with 1 ml of liposomes per 100 g on day 1 as per manufacturer’s instructions. The mice were then injected with 1x10⁵ CFU of S. aureus NewHG (1:1:1 mixture of NewHG Ery^R, Tet^R or Kan^R) on day 2. Blank liposomes were used as a control. Macrophage depletion was confirmed using histology sections of the liver stained with anti-macrophage antibody (rat anti mouse F4/80, AbD Serotec, catalog number MCA497R). For antibody based neutrophil depletion, in vivo anti-ly6G mouse antibody (1A8, BioXcell, catalog number BE0075-1) was used as per the previously published protocol56 (link). The mice were injected with 1.5 mg/mouse of antibody (200 μl per mouse) on day 1 with the mice being injected with 5x10⁵ CFU S. aureus NewHG (1:1:1 mixture of NewHG Ery^R, Tet^R or Kan^R) on day 2. 100 μl of blood was collected via tail bleeding at the time of S. aureus injection and at the end of the experiment via terminal anaesthesia and heart puncture. The blood samples were mixed with 20 μl of Heparin each and then stained with APC Rat Anti-Mouse Ly-6G antibody (BD biosciences, catalog number 560599) according to the BD bioscience protocol. The samples were then processed using the BD LSRII flow cytometer to confirm neutrophil depletion.