backend=biber, style=alphabetic, sorting=ynt ]biblatex 2.6. Cultured Spleen Cells and Immunophenotyping to Flow cytometry for Cell Subsets and Intracellular Cytokines Analysis
Splenic cells (5×10⁶) were cultured in RPMI-1640 for 12 h at 37 °C with 5% CO₂, with or without Soluble Epimastigote Antigen (EPI) stimulation. Brefeldin A (10 µg/mL) was added for the final 4 h. After EDTA treatment and FACS buffer washing, cells were stained for surface markers (CD4, CD8, CD19, NK1.1, MAC3, CD11c) using FITC-conjugated antibodies. After fixation and permeabilization, intracellular cytokines (TNF-α, IFN-γ, IL-4, IL-10) were stained with PerCP, APC, or PE-labeled antibodies. Flow cytometry was performed acquiring 30,000 events/sample on a FACScalibur cytometer. Data were analyzed using FlowJo software v10.0.1.