Cytokine Production Assay for Malaria Peptides

3 × 10⁶ isolated leukocytes were distributed in 96-well plates and resuspended in 100 μl of complete RPMI medium. The immunogenic peptides PbGAP50₄₀₋₄₈ (SQLLNAKYL-NH2; peptides&elephants, Hennigsdorf, Germany) (22 (link)) and PbTRAP_130−138 (SALLNVDNL-NH2; peptides&elephants, Hennigsdorf, Germany) (51 (link)) were used. 50 μl peptide diluted in RPMI (10 g/ml) was incubated for 2 h at 37°C, 5% CO₂. Thereafter, 50 μl of Brefeldin A (1:1,000; eBioscience) was added to block secretion of cytokines, and cells were incubated for additional 4 h. After centrifugation cells were stained for surface markers with CD3e-PeCy7, CD4-BV421™ (clone RM4-5; Biolegend) and CD8-BV570™ antibodies, followed by staining with Pacific Orange-NHS for cell viability. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then permeabilized with Perm/Wash™ buffer (BD Biosciences). Intracellular IFN-γ was stained with Interferon gamma-APC antibody (clone XMG 1.2; eBioscience). Cells were washed in Perm/Wash™ buffer, then resuspended in PBS/1% BSA for acquisition (stopping gate of 20,000 CD8⁺ T cells) with MACS Quant Analyzer 10 and analysis by FlowJo software (51 (link)).