Immune Cell Profiling in Malaria-Infected Mice

Mice infected with PbA were euthanized at 5 dpi. The brains and spleens were removed and processed according to the methodology described by Brant et al. [37 (link)]. Lungs were removed and processed according to the methodology described by Claser et al. [12 (link)]. Purified cells from the brain, lung, and spleen were plated and incubated with brefeldin A (10 μg/mL) (Invitrogen, Waltham, MA, USA) for 3 h at 37 °C in the presence of 5% CO₂. The cells were blocked with Fc Block (antibody CD16/CD32 in PBS/BSA 1%), followed by staining with specific combinations of antibodies for cell surface molecule labeling: CD3 and CD11b (APC-Cy7); CD4 and Ly6C (PE-Cy7); CD8, Ly6G, and SinglecF (BV421); CD25 and CD45 (PerCP-Cy5.5); F4/80 (FITC); CD11c (V500); and isotype controls (all antibodies from BD Biosciences, Franklin Lakes, NJ, USA). For intracellular staining, the following antibodies were added: IFN-γ (Alexa 488), IL-17A, Foxp3 (PE), TNF (PE), and IL-10 (APC). A total of 30,000 cells (events) were acquired using a FACS Canto II cytometer (BD Biosciences) and analyzed using FlowJo software (ver. 10). Our gating strategy is shown schematically in Supplementary Figure S1 (for innate immune cells subset composition) and Supplementary Figure S2 (for adaptative immune cells subset composition).