Legendplex mouse cytokine panel

Supernatants from the skin and cerebral homogenates were assayed using a fluorescence bead-based LEGENDplex Mouse Cytokine Panel (BioLegend, San Diego, CA, USA) as described [9 (link)]. This kit simultaneously quantifies 13 mouse cytokines, including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α. Assays were carried out according to the manufacturer’s instructions. Cytokine levels were detected by flow cytometry using BD-FACSymphony A3 (BD Biosciences, San Jose, CA, USA). Data were analyzed with the LEGENDplex™ software provided in the kit (version 8, BioLegend, San Diego, CA, USA).

Mice were infected with BS parasites by injecting 10⁶ iRBCs of either Py or Pb parasite. Blood was collected from heparin tubes at different time points during the course of infection and plasma was harvested by spinning tubes at 10,000 rpm at 4 °C for 10 min. The samples were immediately stored at −80 °C until further use. The expression of cytokines (IFNγ, IL-6, IL-12p40, IFNα, TNFα, IL-1α, IL-10, IFNβ, IL-1β, IL-12p70, and TGFβ) was determined in bead-based multiplex immunoassay using a customized LEGENDPlex mouse cytokine panel (Biolegend, San Diego, USA). The assay was performed as per manufacturer’s instructions and data was acquired on BD LSR II flow cytometer using BD FACSDiva software (v8.0.1). The data was analyzed using LEGENDplex™ Data Analysis Software, (v2023-02-12, 58444) (BioLegend, USA) as shown in Fig. S3A^{68 (link)}. The standard curve was created for each analyte (R² >0.99) by performing 1:4 dilution (8-points) of mouse custom standard panel (provided in the kit) with starting top concentration 10,000 pg/ml or 50,000 pg/ml (IFNβ). The assay was performed by simultaneously using the samples from two to three independent experiments.